Crispr

IND US 20190345483A1
IN
(19) United States
( 12) Church
Patentet Application
al.
Publication ((4310)) Pub
Pub.. No.: US 2019/0345483
Date :
A1
Nov. 14 , 2019
(54 ) AAV SPLIT CASI GENOME EDITING AND Publication Classification
TRANSCRIPTIONAL REGULATION (51) Int. Ci.
C12N 15/10 (2006.01)
( 71) Applicant: President and Fellows of Harvard C12N 15/86 (2006.01 )
College , Cambridge , MA (US) C12N 15/90 (2006.01)
(72) Inventors: George M. Church , Brookline, MA C12N 9/22 ( 2006.01)
(US); Wei Leong Chew , Boston , MA (52 ) U.S. CI.
(US ) CPC C12N 15/1024 (2013.01 ); C12N 15/86
(2013.01); C12N 2310/20 ( 2017.05) ; C12N
(21) Appl. No.: 16 /099,828 9/22 ( 2013.01); C12N 15/90 (2013.01 )
( 57 ) ABSTRACT
( 22 ) PCT Filed : May 12 , 2017 The invention provides methods of altering a target nucleic
( 86 ) PCT No.: PCT /US17/32362 acid in a cell using the AAV split Cas9 platform . The
methods comprise providing the cell an enzymatically active
$ 371 (c )( 1) , Cas9 and optionally a transcriptional regulator fused thereto
(2 ) Date : Nov. 8 , 2018 and guide RNA having different spacer sequence lengths
wherein the guide RNA directs the enzymatically active
Cas9 and optionally a transcriptional regulator fused thereto
Related U.S. Application Data to either cleave a target nucleic acid or regulate expression
(60 ) Provisional application No. 62 /335,271, filed on May of a target nucleic acid .
12 , 2016 . Specification includes a Sequence Listing.
Split-Cas9 AAV Transduction

N -intein
Cas9N
H
Cas9FL Scarless reconstitution
Cas9FL
Cas9
C - intein 0
Patent Application Publication Nov. 14 , 2019 Sheet 1 of 31 US 2019/0345483 A1
FIG . 1A
-Sp*
Untargebl
100 100 108
!W !W ? 80 Sa *
Sp
) 10
Exons
%
(
1 ofFraction genes
all
)
%
(
40
20
60
pOaRiNrAs inexome 106
104
Sa
Nm
0.1 0 St1
SpSaSt1UN Sa
ds
*
[ *
-100 -10
Closest PAM
-1 20 60 100
Distance between
upstream of TSS (bp) target sites (bp )
FIG . 1B
Split-Cas9 AAV Transduction
N - intein
Cas9N
H
CasgF Scarless reconstitution
1
f Cas9F
C - intein
Cas9 PETTO
??
FIG . 1C
P - 0.84
F = 0.49
C)catsi9viFtLy 120
Avs
%
(
100
80
wahana dana nakon
porno
? 2
ma
Cas9N:Cas90
FIG . 1D
Cas9N : Cas9
1 :0
2: 1
Mf(%u)rteaquteinocy - 1: 1
1 :2
0:1 n.s.
O
1E9 ?? 1E12
AAV titer (vg)
FIG . 1E
100
??
Mfurteaqioncy
Mstn
on
%
(
)
under
• AAV-Cas9
* AAV -Cas9 - VPR
CAAVRNA lysates (ul/well)

FIG . 1F
Mstn
PD -L11
Fst
CD47
FIG . 1G
Pd-11 Fst Cd47
***
40 10 2
co
30
Rexlpratsivoen 20
UT
6
4
2
1
ORNA 1 +
ORNA 2 + +
Non -target gRNAs +
+
Pd - 11 Fst Cd47
**** *** ***
40 8 2
30 6
Rexplratsivoen 20
10
4
2
1
ORNA 1 + + + + + +
ORNA 2 + + + +
Non -target gRNAS
FIG . 1H
R = -0.76 , P < 0.001
Pd- 11 st Cd47
30
20
aF-ctoivlatidon 10
0 ..
20 40 60 80 100
Basal expression percentile
FIG . 2A
)
6
3
FIG . 2B
NLS IntN polya
SMP Cas9NT
2.5 kb
Into NLS polya
SMVP Cas90
2.2 kb
Intc NLS P2A polyA
SMVP Casgo
2.9 kb
GEP
FIG . 2C
CasgN Casgo Cas9N Cas9 -P2A -GFP
20 , 20 ,
P = 0.298 P = 0.118
F = 1,334 15 w
2.429
Acvr2b 5 Acvr2b
20
**** 3
1
:
40:
1
MockCas9F. 20
1
:
04:
1312
: 11
: :
1 31
il7
: :
4
MockCaP2AsgF-GFP
l
Mf(%)urteqauteinocy 15
P0.173
Fra 1.726
P = 0.630
F = 0.768 Indel
Acvr2a
0
Acvr2a THE
Inversion
Insertion
Deletion
20 . P0.984
MockCas9F1 20
04
1
: 13
: :
12:
1 1
:
3140
: :
1
P = 0.735
MOCKCasP2AFilm-GFP
15 ! F = 0.189 15 F 0.611
Mstn Msin
IT 111 MockCasgail.com
MockCasoft
0
:1 2
:
11:
1121
: :
31:
4
P2A-GFP
FIG . 2D
Td5 Td5 TOL TAR Td5
Td3 TOR Td3 TDR Td3
CAG 3x Stop td Tomato C.as9Fb

Td5
TdL
Td3
TOR
.Casgt TAR no ORNA
CAG Notala Cas9S-plit
FIG . JA
ITR NLS IntN polya ITR
SMVP CasON 06-9RNA
2.5 kb
ITR Intc NLS P2A polyA ITR
we s CASI Cas90 GFP
2.2 kb
FIG . 3B
2d 50 6d Mock (60 )
.
FIG . 30
Msta Acvr2b Acvr2a
2.5 P < 0.001 2.5 P < 0.001 20
p < 0.001
P < 0.01 P < 0.01 P < 0.001
2 2
Mf(%)urteqauteinocy 1.5 Mf(%)urteaquteinocy .5 Mf(%)urteaquteinocy 15
10
5
0.5
1E10
vg
AAVlysates noORNA 1E10
vg
AAVlysates noORNA 1E10vgnoCRNA
AAVlysates
FIG . 3D
Td5 + Td3 TIL + TAR 6.7E11 of
1: 2 2:1 1: 1 Cas9C -P2A -GFP
CAG 3x Stop td Tomato
Td5
TdL
Td3
TOR
TdTomato
CAG
GFP
FIG . 4A
AAV9 3xStop
Cas9- gRNAs (gRNAsTdL + TdR )
Mstn
(gRNAsM3+M4)
FIG . 4B
Liver
Heart
Gastrocnemius
10 Brain
Gonad
Mf(%)urteaquteinocy .Diaphragm
--
1
1
1
1
... LLLLL..........
1E0 1E1 1E2 1E3

AAV copies per diploid (vg /dg )
FIG . 4C
Liver Heart Muscle Brain Gonad
Female *****
C+
R
&T
gCas9
-
AAV9
.
M.4
asR9N.gARN?AMsT3a Male
Female ::
Male
FIG . 4D
3
Cd47
value
)
-Logo
-
q
( 2 *
Pd- 11
0 0
-5 5
Log ,( fold -change)
FIG . 4E
2.0x, P < 0.05 1.6x, P < 0.05
200 6 ??? ? ????
150 4
100
50
w
0
Group R 2 R2 ???
AAV9-turboRFP ( 1E11 ) +
mà
AAV9-Cas9C- VPR (2E12 )

AAV9-Cas9N -gRNAsMsn ( 1E12 ) f +
AAV9- Cas9N - ORNAFS' (1E12 )

AAV9-Cas9N- ORNAFSU (3.3E11 )
AAV9-Cas9N -ORNASPA (3.3E11 )
AAV9-Cas9N -ORNASC047( 3.3E11)
FIG . 5A
0.8
0.6
*
0.4
0.2
Mf(%)urteaquteinocy o
Indel
Inversion
Insertion
Deletion
D
LiverHeart BrainGonad
Gastrocnemius i a p h r a g m
FIG . 5B
Locus Protospacer PAM Off-target
mutation ( % )
Mstn on -targetM3 AAGTCTCTCCGGGACCTCTT GGG 02 4
chr 16 : +3906202 AAGGCTCTCCAGGACCTCTT GGG

chr4 :-55206243 AAGTATCTCTGGGACCTCTT CAG
chr13 : +38093999 GAGTCCCTCCGGGAGCTCTT GGG
Mstn on -target M4 GTGCTGCCGCTACCCCCICA CGG 0 2 4

chr2 :-49015581 GTGCTGCAGCTGCCACCTCA GGG Liver
chr5 : +132154281 GTGCAGCCGCTACCACCACA AAG a Heart
O Sequencing error
FIG . 5C
AAVI-GFP - Cre
(2.5E11) Vehicle
td Tomato DAPI td Tomato DAPI
Liver 85 vg/dg
Heart 34 vg/dg
Muscle 64 vg / dg
Brain 2.4 vg /dg
Testis < 1 vg/dg

FIG . 5D
AAVI-GFP (2E12 )
AAV9-mCherry (2E12 ) Vehicle
Co
EGFP mCherry DAPI transduced EGEP mCherry DAPI
Liver
Heart
Muscle
Brain
Testis
FIG . 5E
100
ning M4 only
80
Fofm(%)ruatcanitosn 60 a M3 only
40
M3+M4
Precise
20 excision
Diaphragm
Liver Heart
FIG . 5F
1E4 *** *** ***
1E3
AAV
cper
d(
vg
dg
/
)oiplioeisd 1E2
151
para
..me
1E0
Dia
LiverHeart BrainGonad
lulu
Gastrocnemius phr agm
FIG . 5G
1E2
c(
AAV
dvg
per
)ioplioeisd
dg
/
M
1E1
1E0
LiverHeart
FIG . 5H
Gastrocnemius
10 Liver
Heart
Gastrocnemius
Mfu(%)rteaqtueinocy Brain
4E12 vg
1E0 151 152 1E3

AAV copies per diploid
FIG . 6
Liver Heart Muscle Brain Testis
AAV9-Cas9 -gRNASTEL + TOR

3x Stoo totoniato
AAV9-Cas9 - gRNASTUSTA3
CAG 3x Stop
AAV9-Cas9 -gRNAS$ 3***

AAV9 - Cas9 -gRNASTOLETAR
CAG 3x Stop to Tomato
AAV9.Cas9.gRNAsTS+T81.
CAG 3x Stop to Tomato
AAV9 -Cas9-9RNAS 1034TR

CAG 3X Stop IdTomato .
Vehicle
FIG . 7A
TdTomato GFP DAPI
TOLAR
AAV9-Cas9GRNASTI
g+Cas9
M4-AAV9RNASM3
FIG . 7B
Td Tomato GFP DAPI
TdR
gCas9
+-AAV9
RNASTAL
000
000
FIG . 7C
ToTomato DAPI
TILATOR
AAV9-Cas9gRNAST
304
AAV9
Cas9
-
g*
M
RNASM3
..
FIG . 8A
A V 9
PlasmCiads9FL Mock
Split
-Cas9
Cas9FL
Cas9N : Casgc
GAPDH
Cas9FL reconstitution ( % ) 47 51
Cas9FGAPDH (AU ) 1.3 5.2 13 12 -
FIG . 8B
*
18
)
n
x106
(
l o
y
cCinoeulnt 10 d
m
14e
p s
h
6
Cas9 2
Promoter + polyA V V
Electroporated
AAV VV
Patent Application Publication Nov. 14, 2019 Sheet 23 of 31 US 2019/0345483 A1
FIG . 8C
0.06
P <0.001
P < 0.001
M-Horisrtna isnmdlaerixty 0.04

0.02 29
Cas9VectoVrehicle
FIG . 8D
P < 0.01
Clontypic a(%)bundance 0.6

IN S
0.2
0
Media +Cas9
FIG . SE
X Gene
fragmentation
M13 phage
Serum library
antibodies
Mapped epitope
Immunoprecipitation
Sequencing
FIG . 8F
Cas9
epitopes +
REC1 PI
FIG . 8G
AAV9
epitopes 10
Counts 5 -log
,
P
(
VP1 adj
VP2
VP3
FIG . SH
que
(AU)persi tency
bMultoandt
?? k ?????????????????????????????????
Antigenicity
FIG . 81
1.5
Mt(AU)urotpainstm
Global tropism
0.5 ( except liver)
Hepatotropism
0
Antigenicity
FIG.9A
7A9 (mAb )
> 10
Guide-IT (pAb )
bG15 (PAD )
bS18 (pAbEEE
)
Wwwwwwwwwwww
)-og.Ped
bD20 (pAb) C
REC1 PI
FIG . 9B
Cas9FL
1126 -WD auto Cas9
Cas9FL 1135DE
- A1126-1135 Mock
Td3
+
Td5
0
TAR
+
TOL
FIG . 9C
IgG2a IgM
: 3
f(AU)luorescence
nForImalAizXed 20
kan
ke
za
szerepe om
0 0
1E1
Vehicle 4.1E12 1 E1
Vehicle 4.1E12 1 E 1
Vehicle 4.1E12
FIG.9D
1.2
1.0
sarea
ratio(AU)urface 0.8
-aScolesvibnlte 0.6
Median
0 1 2 3 4 5 6 7 8
Antigenicity
180 °
+
FIG . 10A Perforin

Pertorini DAPI
R O
2 09
-VPROA RV9N.CAasS9*AAV9-turbORFP a
2
8 3
Q S
D.
AAV9
VPR
Casg
-
ORNAS
!
t*AAV9-urDORF D
B D
0
23
BE
A-tournbVolR9Ey 23
D
D
0
WWW
Patent Application Publication Nov. 14. 2019 Sheet 30 of 31 US 20190345483 A1
FIG . 10B Perforin

Pertorin
Casol
Casoriai
GEP
...”
GFP
FRS
F
FIG . 10C
n.s., P = 0.35
Centralynucleatd )
%
(
myofibers 4
2
0
n.s., P > 0.5
*****
AAV9-Cas9N- gRNAsset 1
AAV9 -Cas9N - gRNAsset2 +
AAV9- Cas9C-VPR
AAV9 -turbORFP
FIG . 10D
Centrally
nucleated
Non- centrally
nucleated
***
P = 0.37
50
sieqyosu
)
%
(
nCuecltraedy 30
20
0
*
Electroporation VIN
Cas9
GFP
FK506 V
US 2019/0345483 Al Nov. 14 , 2019
AAV SPLIT CASI GENOME EDITING AND Nature methods 10 (10 ), 973 ( 2013 ); L. A. Gilbert, M. H.
TRANSCRIPTIONAL REGULATION Larson, L. Morsut et al., Cell 154 (2 ), 442 ( 2013 ); P.Mali ,
K. M.Esvelt, and G.M. Church , Nature methods 10 ( 10 ),
RELATED APPLICATION DATA 957 (2013 ); and K. M. Esvelt, P. Mali , J. L. Braff et al.,
[0001 ] This application claims priority to U.S. Provisional Nature methods 10 (11 ), 1116 ( 2013 ).
Application No. 62/ 335,271 filed on May 12 , 2016 , which is [0004 ] The CRISPR - Cas9 system enables facile genetic
hereby incorporated by reference in its entirety for all and epigenetic manipulations (Mali, P. et al. RNA -guided
purposes. human genome engineering via Cas9. Science 339, 823-826 ,
doi:10.1126 / science.1232033 ( 2013 ); Cong, L. et al. Multi
STATEMENT OF GOVERNMENT INTERESTS plex genome engineering using CRISPR / Cas systems. Sci
[0002 ] This invention was made with government support ence 339 , 819-823 , doi: 10.1126 /science.1231143 (2013 );
under Grant No. P50 HG005550 awarded by National
Jinek , M. et al. A programmable dual- RNA - guided DNA
endonuclease in adaptive bacterial immunity . Science 337 ,
Institutes of Health . The government has certain rights in the 816-821, doi: 10.1126 /science.1225829 ( 2012 )), and its sim
invention . plicity and robustness suggest that personalized genetic
BACKGROUND
therapeutics may be within reach . As CRISPR -Cas9
approaches the clinic, efficacy and safety for the patient
[ 0003 ] The CRISPR type II system is a recent develop would be of paramount importance. Extensive efforts have
ment that has been efficiently utilized in a broad spectrum of been employed to deliver CRISPR -Cas9 with adeno -asso
species. See Friedland , A. E., et al., Heritable genome ciated viruses (AAVs). AAVs are prevalent and serologically
editing in C. elegans via a CRISPR - Cas9 system . Nat compatible with a large fraction of the human population
Methods, 2013. 10 (8 ): p . 741-3 , Mali , P., et al.,RNA - guided (Gao , G et al. Clades of Adeno-associated viruses are widely
human genome engineering via Cas9 . Science , 2013. 339 disseminated in human tissues. Journal of virology 78,
(6121): p . 823-6 , Hwang , W X., et al., Efficient genome 6381-6388 , doi: 10.1128 / JVI.78.12.6381-6388.2004 (2004 );
editing in zebrafish using a CRISPR -Cas system . Nat Bio Boutin , S. et al. Prevalence of serum IgG and neutralizing
technol, 2013 , Jiang, W., et al., RNA - guided editing of factors against adeno -associated virus (AAV ) types 1, 2 , 5,
bacterial genomes using CRISPR - Cas systems. Nat Biotech 6 , 8 , and 9 in the healthy population : implications for gene
nol, 2013 , Jinek , M., et al., RNA-programmed genome therapy using AAV vectors . Hum Gene Ther 21, 704-712 ,
editing in human cells . elife , 2013. 2: p . e00471, Cong, L., doi:10.1089 /hum.2009.182 (2010 )) and are generally not
et al., Multiplex genome engineering using CRISPR / Cas considered to be pathogenic . Furthermore, AAVs allow both
systems. Science, 2013. 339 (6121): p . 819-23 , Yin , H., et al., programmable tissue- tropism and systemic delivery (Zin
Genome editing with Cas9 in adult mice corrects a disease carelli, C., Soltys , S., Rengo , G & Rabinowitz , J. E. Analysis
mutation and phenotype . Nat Biotechnol, 2014. 32 (6 ): p . of AAV serotypes 1-9 mediated gene expression and tropism
551-3 . CRISPR is particularly customizable because the in mice after systemic injection . Mol Ther 16 , 1073-1080 ,
active form consists of an invariant Cas9 protein and an doi: 10.1038 /mt.2008.76 ( 2008 )). The preclinical promise of
easily programmable guide RNA (ORNA ). See Jinek , M., et AAV -CRISPR -Cas9 by correcting genetic defects in mice
al., A programmable dual-RNA - guided DNA endonuclease has been described (Ran , F. A. et al. In vivo genome editing
in adaptive bacterial immunity. Science, 2012. 337 (6096 ): p . using Staphylococcus aureus Cas9 . Nature 520 , 186-191,
816-21 . Of the various CRISPR orthologs , the Streptococcus doi:10.1038 /nature14299 (2015) ; Nelson, C. E. et al. In vivo
pyogenes (Sp ) CRISPR is the most well - characterized and genome editing improvesmuscle function in a mouse model
widely used . The Cas9 -gRNA complex first probes DNA for of Duchenne muscular dystrophy. Science 351, 403-407 ,
the protospacer-adjacent motif (PAM ) sequence (NGG for doi: 10.1126 /science.aad5143 ( 2016 ); Tabebordbar,M. et al.
Sp Cas9 ), after which Watson -Crick base -pairing between In vivo gene editing in dystrophic mouse muscle and muscle
the gRNA and targetDNA proceeds in a ratchet mechanism stem cells . Science 351, 407-411 , doi:10.1126 / science.
to form an R -loop . Following formation of a ternary com aad5177 (2016 ); Long, C. et al. Postnatal genome editing
plex of Cas9 , gRNA , and target DNA , the Cas9 protein partially restores dystrophin expression in a mouse model of
generates two nicks in the target DNA , creating a blunt muscular dystrophy . Science 351 , 400-403 , doi: 10.1126 /
double -strand break (DSB ) that is predominantly repaired by science.aad5725 ( 2016 ); Yang , Y. et al. A dual AAV system
the non-homologous end joining (NHEJ) pathway or, to a enables the Cas9 -mediated correction of a metabolic liver
lesser extent, template -directed homologous recombination disease in newborn mice . Nature biotechnology, doi:10 .
(HR ). CRISPR methods are disclosed in U.S. Pat. Nos. 1038 /nbt.3469 (2016 ); Yin , H. et al. Therapeutic genome
9,023,649 and 8,697,359. See also , Fu et al., Nature Bio editing by combined viral and non -viral delivery of CRISPR
technology, Vol. 32 , Number 3 , pp . 279-284 (2014 ). Addi system components in vivo . Nature biotechnology , doi: 10 .
tional references describing CRISPR -Cas9 systems includ 1038 /nbt.3471 ( 2016 )) .
ing nuclease null variants (dCas9 ) and nuclease null variants [0005 ] Further application of AAV- CRISPR - Cas9 for
functionalized with effector domains such as transcriptional modulating postnatal chromatin status or gene expression
activation domains or repression domains include J. D. would vest profound biological control, particularly in treat
Sander and J. K. Joung , Nature biotechnology 32 (4 ), 347 ing diseases resulting from epigenetic alterations irresolv
( 2014 ); P. D.Hsu , E. S. Lander, and F. Zhang, Cell 157 (6 ), able by genome- editing . However, this ability has yet to be
1262 (2014 ) ; L. S. Qi,M.H.Larson, L. A.Gilbert et al., Cell realized , in part because the large Cas9 transgenes leave
152 (5 ), 1173 (2013 ); P. Mali , J. Aach , P. B. Stranges et al ., little space for additional function -conferring elements
Nature biotechnology 31 (9 ), 833 ( 2013 );M.L. Maeder, S. within current designs (AAV payload limit s4.7 kb ) (Ran , F.
J. Linder, V. M. Cascio et al., Nature methods 10 (10 ), 977 A. et al. In vivo genome editing using Staphylococcus
(2013) ; P. Perez - Pinera, D. D. Kocak , C. M. Vockley et al., aureus Cas9 . Nature 520 , 186-191, doi: 10.1038 /na
US 2019/0345483 A1 Nov. 14 , 2019
2
ture14299 ( 2015 ) ; Nelson, C. E. et al. In vivo genome binding protein to the double stranded DNA target sequence
editing improves muscle function in a mouse model of for binding thereto . This aspect of the present disclosure
Duchenne muscular dystrophy. Science 351, 403-407, doi: may be referred to as co -localization of the RNA and DNA
10.1126 /science.aad5143 (2016 ); Tabebordbar, M. et al. In binding protein to or with the double stranded DNA .
vivo gene editing in dystrophic mouse muscle and muscle [0008 ] DNA binding proteins within the scope of the
stem cells . Science 351, 407-411 , doi: 10.1126 / science . present disclosure may include those which create a double
aad5177 (2016 ); Long, C. et al. Postnatal genome editing stranded break (which may be referred to as a DNA binding
partially restores dystrophin expression in a mouse model of protein nuclease), those which create a single stranded break
muscular dystrophy. Science 351, 400-403 , doi: 10.1126 / (referred to as a DNA binding protein nickase ) or those
science.aad5725 ( 2016 ); Yang , Y. et al. A dual AAV system which have no nuclease activity (referred to as a nuclease
enables the Cas9 -mediated correction of a metabolic liver null DNA binding protein ) but otherwise bind to target
disease in newborn mice. Nature biotechnology , doi:10 . DNA. In this manner, a DNA binding protein -guide RNA
1038 /nbt.3469 (2016 ); Yin , H. et al. Therapeutic genome complex may be used to create a double stranded break at a
editing by combined viral and non -viral delivery of CRISPR target DNA site, to create a single stranded break at a target
system components in vivo . Nature biotechnology , doi:10 . DNA site or to localize a transcriptional regulator or func
1038 /nbt.3471 (2016 ); Senis, E. et al. CRISPR /Cas9 -medi tional group , function -conferring protein or domain , which
ated genome engineering: an adeno - associated viral (AAV ) may be expressed by the cell , at a target DNA site so as to
vector toolbox . Biotechnology journal 9 , 1402-1412 , doi: regulate expression of target DNA . According to certain
10.1002 /biot.201400046 (2014 ); Swiech , L. et al. In vivo aspects, the foreign nucleic acid sequence may encode one
interrogation of gene function in the mammalian brain using or more of a DNA binding protein nuclease, a DNA binding
CRISPR - Cas9. Nature biotechnology 33 , 102-106 , doi: 10 . protein nickase or a nuclease null DNA binding protein . The
1038 /nbt.3055 ( 2015 )). This obstacle is exacerbated with the foreign nucleic acid sequence may also encode one or more
most widely used , but larger, Streptococcus pyogenes Cas9 transcriptional regulator or functional group , function - con
( SpCas9 , 4.2 kb ), which makes packaging of even the ferring proteins or domains or one or more donor nucleic
minimum functional cassette extremely challenging (Long , acid sequences that are intended to be inserted into the
C. et al. Postnatal genome editing partially restores dystro genomic DNA. According to one aspect, the foreign nucleic
phin expression in a mouse model of muscular dystrophy. acid sequence encoding an RNA guided enzymatically
Science 351, 400-403 , doi:10.1126 / science.aad5725 ( 2016 ); active DNA binding protein further encodes the transcrip
Senis, E. et al. CRISPR / Cas9 -mediated genome engineer tional regulator or functional group, function -conferring
ing: an adeno -associated viral (AAV ) vector toolbox . Bio protein or domain fused to the RNA guided enzymatically
technology journal 9, 1402-1412 , doi: 10.1002/ biot . active DNA binding protein .
201400046 (2014 ); Swiech , L. et al. In vivo interrogation of [0009] Accordingly, expression of a foreign nucleic acid
gene function in the mammalian brain using CRISPR - Cas9. sequence by a cell may result in a double stranded break , a
Nature biotechnology 33 , 102-106 , doi: 10.1038 /nbt.3055 single stranded break and /or transcriptional activation or
( 2015) ) repression of the genomic DNA . Donor DNA may be
SUMMARY inserted at the break site by cell mechanisms such as
homologous recombination or nonhomologous end joining .
[0006 ] Aspects of the present disclosure are directed to the It is to be understood that expression of a foreign nucleic
use of split Cas9 to perform CRISPR -based methods in cells . acid sequence as described herein may result in a plurality
According to one aspect, two ormore portions or segments of double stranded breaks or single stranded breaks at
of a Cas9 are provided to a cell, such as by being expressed various locations along target genomic DNA , including one
from corresponding nucleic acids introduced into the cell. or more or a plurality of gene sequences, as desired .
The two or more portions are combined within the cell to [0010 ] Aspects of the present disclosure are directed to
form the Cas9 which has an ability to colocalize with guide methods of using an enzymatically active Cas9 , such as a
RNA at a target nucleic acid . It is to be understood that the Cas9 nuclease or nickase , optionally having a functional
Cas9 may have one ormore modifications from a full length group attached thereto , and a guide RNA which is used to
Cas9 known to those of skill in the art, yet still retain or have guide the enzymatically active Cas9 with the functional
the capability of colocalizing with guide RNA at a target group attached thereto to a target nucleic acid . According to
nucleic acid . Accordingly , the two or more portions or one aspect, when a functional group is attached to the
segments, when joined together, need only produce or result enzymatically active Cas9, the functional group is directed
in a Cas9 which has an ability to colocalize with guide RNA to a targetnucleic acid to perform the desired function on the
at a target nucleic acid . target nucleic acid , such as transcriptional regulation . Also ,
[0007] According to certain general aspects, when a for it is to be understood that transcriptional regulation can also
eign nucleic acid sequence or sequences are expressed by be accomplished according to methods described herein
the cell , the two or more portions or segments of an RNA where an enzymatically active Cas9 is used without any
guided DNA binding protein , such as Cas9, are produced attached functional group and transcriptional regulation is
and joined together to produce the RNA guided DNA accomplished by inhibition of transcription due to the Cas9
binding protein , such as Cas9 . When a foreign nucleic acid forming a complex at the target nucleic acid and without
sequence or sequences are expressed by the cell, one or more cutting the target nucleic acid . According to one aspect, the
or a plurality of guide RNAs are produced . The RNA guided guide RNA includes a truncated spacer sequence having a
DNA binding protein , such as Cas9 , and a guide RNA length sufficient to bind to a target nucleic acid and form a
produces a complex of the RNA guided DNA binding complex with the enzymatically active Cas9 optionally with
protein , the guide RNA and a double stranded DNA target the functional group attached thereto , but insufficient for the
sequence. In this aspect, the RNA is said to guide the DNA enzymatically active Cas9 to function to cut or nick the
US 2019/0345483 A1 Nov. 14 , 2019
3
target nucleic acid. Without wishing to be bound by scien therefore can regulate gene expression without using a
tific theory , based on the length of the spacer sequence of the separate transcription regulator functional group .
guide RNA , the endonucleolytic activity of the enzymati
cally active Case is blocked or prevented or otherwise [0014 ] Aspects of the present disclosure are directed to
inhibited , and the otherwise enzymatically active Cas9 is programmable genome editing and use of a functional
effectively rendered a nuclease null Cas9 . According to this group , such as a transcriptional regulator, using the same
aspect, the functional group when attached to the enzymati species of enzymatically active Cas9 having the functional
cally active Cas9 performs the desired function of the group attached thereto . Methods described herein are
functional group , as the enzymatically active Cas9 nuclease directed to the use of a single species of enzymatically active
does not function to cut or nick the target nucleic acid . Cas9 having a transcriptional regulator attached thereto
Without wishing to be bound by any scientific theory , the which can be simultaneously used for genome editing of
enzymatically active Cas9 optionally with the functional target nucleic acids and transcriptional regulation of genes,
group attached thereto forms a co - localization complex with based on the spacer sequence length of the particular guide
the guide RNA and the target nucleic acid , however, the RNA . The length of the guide RNA spacer sequence deter
length of the truncated spacer sequence of the guide RNA mines the ability of the enzymatically active Cas9 species
results in an inability of the Cas9 to cleave the target nucleic having a functional group (such as a transcriptional regula
acid substrate . tor ) attached thereto to either (1) function to deliver the
[0011] According to another aspect, the presentdisclosure functional group to a target nucleic acid so that the func
are directed to methods of using a enzymatically active tional group can perform its desired function or (2 ) function
Cas9 , such as a Cas9 nuclease or nickase , optionally having as an enzyme to cut or nick a target nucleic acid .
a functional group attached thereto and a guide RNA with a
spacer sequence having a length sufficient to bind to a target [0015 ] According to certain aspects, the enzymatically
nucleic acid and to form a complex with the enzymatically active Cas9 optionally having a functional group attached
active Cas9 optionally with the functional group attached thereto is present within a cell and two or more guide RNAs
thereto, and sufficient to allow the enzymatically active Cas9 are provided to a cell in series or simultaneously wherein
to function as a nuclease or nickase with respect to the target each guide RNA is designed to complex with the enzymati
nucleic acid . According to one aspect, the functional group cally active Cas9 optionally having a functional group
when optionally attached to the enzymatically active Cas9 attached thereto at respective target nucleic acid sites or
does not perform the desired function of the functional sequences. Each guide RNA has a spacer sequence length
group , as the target nucleic acid is either cut or nicked by the that determines whether the enzymatically active Cas9
enzymatically active Cas9 . optionally having a functional group attached thereto will
[0012] Aspects of the present disclosure are directed to function as either an enzyme to cut or nick a nucleic acid or
methods of using a enzymatically active Cas9 , such as a as a nuclease null Cas9 to form a complex at the target
Cas9 nuclease or nickase, optionally having a functional nucleic acid and deliver the functional group if present to a
group attached thereto , a first guide RNA with a spacer target nucleic acid so that the functional group may perform
sequence having a length sufficient to bind to a first target its function on a target nucleic acid . In this manner, enzy
nucleic acid and form a complex with the enzymatically matically active Cas9 optionally having a functional group
active Cas9 optionally having the functional group attached attached thereto may first be used to cut or nick a nucleic
thereto , but insufficient to allow the enzymatically active acid and then be used to deliver a functional group if present
Cas9 to function as a nuclease or nickase with ect to the to a nucleic acid sequence so that the functional group may
first target nucleic acid , and a second guide RNA with a perform the function or vice versa . According to one aspect,
spacer sequence having a length sufficient to bind to a a plurality of guide RNAs may be used to target the
second target nucleic acid and form a complex with the enzymatically active Cas9 optionally having a functional
enzymatically active Cas9 optionally having the functional group attached thereto , such as a single species of an
group attached thereto , such as a Cas9 nuclease or nickase , enzymatically active Cas9 optionally having a functional
and sufficient to allow the enzymatically active Cas9 to group attached thereto, to a plurality of different target
function as a nuclease or nickase with respect to the second nucleic acid sites to perform either cutting or nicking or
target nucleic acid . According to this aspect, the enzymati functional group delivery .
cally active Cas9 when complexed with the first guide RNA [0016 ] When the enzymatically active Cas9 optionally
at the first target nucleic acid will function as a nuclease null having a functional group attached thereto is used for cutting
Cas9 to deliver a functional group if present to the first target or nicking a target nucleic acid , methods described herein
nucleic acid and the enzymatically active Cas9 when com contemplate the use of one or more donor nucleic acids that
plexed with the second guide RNA at the second target may be inserted into one or more cut or nick sites through
nucleic acid will also function as a nuclease or nickase to homologous recombination or nonhomologous end joining .
either cut or nick the second target nucleic acid . Accordingly,methods described herein are directed to meth
[0013 ] Aspects of the present disclosure are directed to ods of genome editing using the enzymatically active Cas9
programmable genome editing as an enzymatically active optionally having a functional group attached thereto and
Cas9 can be used to cut or nick a target nucleic acid by also methods of targeting a functional group when present to
selection of a first guide RNA sequence and the same Cas9 a target nucleic acid to perform the function of the functional
can be effectively rendered nuclease null by selection of a group using the enzymatically active Cas9 having a func
second guide RNA sequence which allows the Cas9 to tional group attached thereto . One of skill will readily
complex at the target nucleic acid sequence but not cut or understand that the utility of the enzymatically active Cas9
nick the target nucleic acid sequence . Such complex forma optionally having a functional group attached thereto is
tion can have an inhibitory effect on transcription and determined by the spacer sequence length of the guide RNA
US 2019/0345483 A1 Nov. 14 , 2019
4
and whether the guide RNA has a spacer sequence length cell expresses the first portion of the Cas9 protein , the gRNA
that facilitates enzymatic activity of the enzymatically active and the second portion of the Cas9 protein or the second
Cas9 or not. portion of the Cas9 and the transcriptional regulator fusion
[0017 ] According to one aspect, a functional group may be protein , wherein the first portion of the Cas9 protein and the
any desired functional group as known to those of skill in the second portion of the Cas9 protein , or the first portion of the
art. An exemplary functional group may be an effector Cas9 protein and the second portion of the Cas9 and the
domain , such as a transcriptional activator or transcriptional transcriptional regulator fusion protein are joined together to
repressor, or a detectable group , such as fluorescent protein , form the Cas9 protein or the Cas9 fusion protein , and
or a binding functional group , such as an aptamer or a wherein the gRNA and the Cas9 protein , or the gRNA and
protein -protein binding domain , which can be used to bind the Cas9 fusion protein form a co -localization complex with
to a desired functional group or a nuclear localization signal, the target nucleic acid and alter the expression of the target
which can be used to deliver the Cas9 to a nucleus. nucleic acid .
[0018 ] According to certain aspects, a guide RNA that [0022 ] According to still another aspect, the present dis
allows enzymatic activity of the enzymatically active Cas9 closure provides a method of modulating a target gene
having a functional group attached thereto includes a spacer expression in a cell including providing to the cell a first
sequence having an exemplary nucleotide length of between recombinant adeno-associated virus comprising a first
about 25 and about 15 nucleotides , such as between about 20 nucleic acid encoding an N - terminal portion of the Cas9
and about 16 nucleotides. According to certain aspects , a protein (Casoy) and a gRNA , providing to the cell a second
guide RNA that inhibits enzymatic activity of the enzymati recombinant adeno -associated virus comprising a second
cally active Cas9 optionally having a functional group nucleic acid encoding a fusion protein comprising a C -ter
attached thereto includes a spacer sequence having an exem minal portion of the Cas9 protein (Cas99) fused with a
plary nucleotide length of between about 8 and about 16 transcriptional regulator ( TR ),wherein the cell expresses the
nucleotides . According to certain aspects, a guide RNA that Cas9N protein and the Cas9C- TR fusion protein and join
inhibits enzymatic activity of the enzymatically active Cas9 them to form a full length Cas 9FL- TR fusion protein , and
optionally having a functional group attached thereto wherein the cell expresses the gRNA , and the gRNA directs
includes a spacer sequence having an exemplary nucleotide the Cas9FL - TR fusion protein to the target gene and modu
length of between 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 lates target gene expression .
and 20 nucleotides. A truncated spacer sequence has a [0023 ] According to yet another aspect, the present dis
nucleotide length that is shorter than the full length spacer closure provides a method of imaging a target nucleic acid
sequence of the corresponding guide RNA. in a cell including providing to the cell a first recombinant
[0019 ) According to certain aspects, a guide RNA includes adeno -associated virus comprising a first nucleic acid encod
a spacer sequence and a tracr mate sequence forming a ing an N - terminal portion of the Cas9 protein ( Cas9m) and
crRNA , as is known in the art. According to certain aspects , a gRNA, providing to the cell a second recombinant adeno
a tracr sequence , as is known in the art, is also used in the associated virus comprising a second nucleic acid encoding
practice of methods described herein . According to one a fusion protein comprising a C -terminal portion of the Cas9
aspect, the tracr sequence and the crRNA sequence may be protein (Cas99) fused with a fluorescent protein ,wherein the
separate or connected by the linker, as is known in the art. cell expresses the Cas9N protein and the Cas9C fluorescent
[0020 ] According to one aspect, the present disclosure fusion protein and join them to form a full length Cas9FL
provides a method of altering a target nucleic acid in a cell fluorescent fusion protein , and wherein the cell expresses the
including providing to the cell a first nucleic acid encoding ORNA , and the gRNA directs the Cas9FL fluorescent fusion
a first portion of a Cas9 protein and a guide RNA (GRNA ), protein to the target nucleic acid and produces fluorescent
providing to the cell a second nucleic acid encoding a second imaging of the target nucleic acid .
portion of the Cas9 protein and optionally a transcriptional [0024 ] Further features and advantages of certain embodi
regulator, wherein the cell expresses the first portion of the ments of the present invention will become more fully
Cas9 protein , the gRNA and the second portion of the Cas9 apparent in the following description of embodiments and
protein or the second portion of the Cas9 and the transcrip drawings thereof, and from the claims.
tional regulator fusion protein , and wherein the first portion BRIEF DESCRIPTION OF THE DRAWINGS
of the Cas9 protein and the second portion of the Cas9
protein , or the first portion of the Cas9 protein and the [0025] The patent or application file contains at least one
second portion of the Cas9 and the transcriptional regulator drawing executed in color. Copies of this patent or patent
fusion protein are joined together to form the Cas9 protein application publication with color drawing (s) will be pro
or the Cas9 fusion protein , wherein the gRNA and the Cas9 vided by the Office upon request and payment of the
protein , or the gRNA and the Cas9 fusion protein form a necessary fee . The foregoing and other features and advan
co -localization complex with the target nucleic acid and tages of the present embodiments will be more fully under
alter the expression of the target nucleic acid . stood from the following detailed description of illustrative
[0021] According to another aspect, the present disclosure embodiments taken in conjunction with the accompanying
provides a method of altering a target nucleic acid in a cell drawings in which :
of a subject including delivering to the cell of the subject a [0026 ] FIGS. 1A -H depict that split -Cas9 retains full
first nucleic acid encoding a first portion of a Cas9 protein activity of Cas9FL and enables AAV packaging with fusion
and a guide RNA (GRNA ) wherein the first nucleic acid is domains. (FIG . 1A ) SpCas9 (canonical PAM : NGG ) broadly
within a first vector, delivering to the cell of the subject a targets the human exome and transcriptional start sites
second nucleic acid encoding a second portion of the Cas9 ( TSS ), while orthologs suffer from restrictive PAMs (Sa :
protein and optionally a transcriptional regulator wherein the NNGRRT; St1: NNAGAAW ; Nm : NNNNGATT ). Sp * and
second nucleic acid is within a second vector, wherein the Sa * denote engineered Cas9 variants and include non
US 2019/0345483 A1 Nov. 14 , 2019
5
canonical PAMs. (FIG . 1B ) Schematic of split - Cas9 and turboGFP expression , with onset by 1-2 days post - transduc
AAV -Cas9. (FIG . 1C ) Split - Cas9 achieves equivalent edit tion . (FIG . 3C ) Both unpurified AAV-Cas9 - gRNAs -contain
ing frequencies as Cas9F1 (one-way ANOVA ). Each data ing lysates ( 100 ul per well ) or 1E10 (vg, vector genomes)
point depicts meants.e.m . of 6 means (source data in FIG . of chloroform -ammonium sulfate purified AAV - Cas9-gR
2C ). (FIG . 1D ) AAV -Cas9 - gRNAs gene- edited myotubes. At NAs edited the targeted endogenous loci in differentiated
each functional Cas9N :Cas9C, mutation frequency increased C2C12 myotubes. Cas9N: Cas9c ratio of 1:1 was used . Each
with AAV dose (one-way ANOVA ), but began to plateau at dot represents the mutation frequency detected per trans
~ 6 % (n.s., not significant between 1E11 and 1E12). ( FIG . duction per condition (P - values , one - tailed Wilcoxon rank
1E ) AAV- Cas9- gRNAs (black ) edited the Mstn gene in sum against no - gRNA controls, Bonferroni corrected). Red
GC -1 spermatogonial cells, while AAV -Cas9-VPR -gRNAs lines denote means = s.e.m . (FIG . 3D ) Transduction of Ai9
(cyan ) exhibited reduced endonucleolytic activity (Cas9N : tail -tip fibroblasts with 1E12 (total vg) ofAAV -Cas9- gRNAs
Cas9 " , 1: 1) (n -way ANOVA ). (FIG . 1F ) Schematic of targeting the 3xStop cassette induced excision -dependent
genome-editing and transcriptional regulation within a fluorescence activation (n = 2 transductions ).gRNA pairs and
single system . A nuclease -active Cas9 is fused to a tran Cas9N :Cas9C - P2A -turboGFP ratios are indicated . Td5 and
scriptional activator domain . Cas9 -mediated endonucle TdL target 5 ' of 3xStop ; Td3 and TdR target 3 of 3xStop .
olytic DNA cleavage is programmed with a full- length TdTomato was not observed in negative controls transduced
ORNA , whereas Cas9 -mediated transcriptional activation is with 6.7E11 (total vg )ofCas9C -P2A - turboGFP only . Images
programmed with a truncated ORNA . (FIG . 1G ) AAV - Cas9 were taken 7 days post-transduction . Scale bars, 500 um .
VPR - gRNAs upregulated target genes, as assessed by qRT [0029 ] FIGS. 4A -D depict postnatal genome-editing with
PCR ( top , GC -1 cells; bottom , C2C12 myotubes; one -way AAV9 -Cas9 - gRNAs and transcriptional activation with
ANOVA ). (FIG . 1H ) Transcriptional activation levels cor AAV9-Cas9 - VPR -gRNAs. (FIG . 4A ) AAV9 - Cas9- gRNAs
relate inversely with basal gene expression levels. Data from targeting the endogenous Mstn gene or the 3xStop cassette
GC - 1 cells in red , data from C2C12 myotubes in black ; in neonatal mice. ( FIG . 4B ) Mutation frequency correlates
closed dots denote with single - gRNA, and open circles with AAV transduction efficiency (Pearson's R = 0.73 , Spear
denote with dual-gRNAs. * , P < 0.05, *** , P < 0.001, ANOVA man's p = 0.74 , P < 0.05 ) (n = 4 mice , 4E12 of AAV9-Cas9 .
followed by Holm - Šidák test. Error bars denote s.e.m. gRNAsM3+4). Horizontal dashed line = sequencing error rate ;
[0027 ] FIGS. 2A -D depict that split-Cas9 retains full vertical dashed line = qPCR false positive rate . Error bars
biological activity of full -length Cas9. (FIG . 2A ) SpCas9 denote s.e.m.for sequencing and qPCR replicates. (FIG . 4C )
consists of a bi- lobed structure (PDB IDs: 4008 and 4CMP ) AAV9-Cas9- gRNAs TdL + TdR -edited tdTomato + cells were
(Nishimasu , H. et al. Crystal structure of Cas9 in complex detected in multiple organs of Ai9 mice (2 upper rows) (n = 3
with guide RNA and target DNA . Cell 156 , 935-949, doi: mice at 4E12 ), and absent in Ai9 mice injected with AAV9
10.1016 /j.cell.2014.02.001 (2014 ); Jinek , M. et al. Struc Cas9 -gRNAsM3+ M4 (2 lower rows) (n = 4 mice at 4E12 ).
tures of Cas9 endonucleases reveal RNA -mediated confor Gray = tdTomato . Scale bar, 5 mm . (FIG . 4D ) AAV9-Cas9 .
mational activation . Science 343, 1247997 , doi: 10.1126 / VPR -gRNAs activated the target Pd - 11 and Cd47 genes in
science.1247997 (2014 )), with the N- and C -termini of the adult mice (FDR = 0.05 ). Volcano plot shows total mRNA
disordered linker indicated . Cas9 is shown bound to the sequencing of the samemuscle samples used for qRT-PCR
GRNA (red ribbon ) and target DNA (blue ribbon). (FIG . 2B ) in FIG .4E (n = 3 mice per condition ). (FIG . 4E ) AAV9-Cas9
Schematic of plasmids encoding split -Cas9 . VPR -gRNAs activated the target Pd - 11 and Cd47 genes in
SMVP = promoter; IntN /IntC = split - inteins; NLS = nuclear adult skeletal muscles, as assessed by qRT-PCR and calcu
localization signal; polyA = SV40 polyadenylation signal. lated as 2-4AACt (n = 3 mice per group ). Fold -change in gene
(FIG . 2C ) Split- Cas9 was tested against full -length Cas9 expression was quantified between AAV9 - Cas9 - VPR -GR
(Cas9F ) on three endogenous genes, with or without co NAs-treated samples that differed only in the gRNA spacer
translating P2A -turboGFP, by transfecting C2C12 cells with sequences (one -tailed t-test ). Samples treated with AAVI
equal total mass amounts of Cas9 plasmids. Mutation fre Cas9 - VPR - gRNAs and AAV9-turboRFP showed transcrip
quencies induced by split - Cas9 and Cas9FL are not signifi tional alterations against samples treated with AAV9-tur
cantly different across all three genes (one -way ANOVA ) boRFP only , due to immunity -associated transcriptome
(400 ng of total Cas9 plasmids and 400 ng of total gRNAs perturbation . AAV-Cas91 -gRNAs:AAV-Cas9C -VPR ratio of
plasmids). Left panels: Cas9 without P2A - turboGFP (n = 3 1 : 1 was used in all experiments. Error bars denote s.e.m.
independent transfections); Right panels: Cas9 with P2A [0030 ] FIGS. 5A -H depict that systemically delivered
turboGFP (n = 2 independent transfections ). Error bars AAV9 -Cas9 -gRNAs genetically modify multiple organs,
denote s.e.m. (FIG . 2D ) Split-Cas9 targets Ai9 fibroblasts with editing frequency reflecting viral transduction effi
equivalently to Cas9FL, activating tdTomato fluorescence by ciency . (FIG . 5A ) Deep -sequencing of tissues indicates
excision of the 3xStop terminators cassette. Sparse tdTo mean Mstn gene- targeting rates ranging from 7.8 % to 0.25 %
mato + cells were observed with single- gRNA , or paired (n = 4 mice , 4E12 of AAV9 -Cas9 -gRNASM3+ M4) (* , P < 0.05 ,
gRNAs both targeting one side of 3xStop (n = 2). Td5 and Wilcoxon rank -sum , Bonferroni corrected ). Error bars
TdL target 5' of 3xStop ; Td3 and TdR target 3 ' of 3xStop . denote s.e.m. Black dashed line denotes sequencing error.
Gray = dTomato . Scale bar, 200 um . (FIG . 5B ) Putative off-target sites were assessed by deep
[0028 ] FIGS. 3A - D depict that transduction ofAAV -Cas9 sequencing. The bona fide off -target locus (chr16 :+3906202)
gRNAs directs gene -editing in differentiated myotubes and contains two mismatches (in red ) against the on -target
M3 +M4.
tail-tip fibroblasts . (FIG . 3A ) Schematic of AAV-Cas9 -gR sequence . (n = 4 mice , 4E12 of AAV9- Cas9- gRNASTdL
NAs. ITR = AAV inverted terminal repeat; SMVP and and n = 2 control mice , 4E12 of AAV9 -Cas9 -gRNAs? + TAR
CASI = promoters; IntN /IntC = split -inteins; NLS = nuclear for determination of baseline sequencing error rates). (FIG .
localization signal; polyA = SV40 polyadenylation signal. 5C ) Recombinase -activated tdTomato fluorescence by
( FIG . 3B ) Epifluorescence time course of AAV-Cas9C-P2A AAV9-GFP -Cre (n = 2 , 2.5E11 ). Mean vg /dg shown. All
US 2019/0345483 A1 Nov. 14 , 2019
6
examined cells within the liver, heart and muscle recom from Cas9 -exposed animals (top, n = 4 electroporated ; bot
bined , indicating ~ 100 % transduction efficiency within these tom , n = 4 AAV9- delivered ). Immunodominant epitopes
organs. Within the testis , absence of tdTomato + cells in the reside in REC1 and PI domains (vertical dotted lines ), and
germline-residing seminiferous tubules argues against represented on Cas9 structure (PDB ID : 4CMP). Red ,
AAV9 transmission to the male germline. (FIG . 5D ) Dual immunodominant epitopes ; Black , private epitopes ; Cyan ,
AAVIS co -transduced multiple organs (n = 2 , 2E12 each of REC1; Pink , PI. P -values from Wald test, Benjamini-Hoch
AAVO-GFP and AAV9-mCherry). (FIG . 5E ) Triple -AAVIS berg adjusted for FDR = 0.1 . (FIG . 8G ) Capsid epitopes from
co - transduction generated double edits on the same chro AAV9 -exposed animals (n = 8 ). Counts denote number of
matin (n = 4 mice co - injected with 2E12 of AAV9-Cas9C animals with capsid - specific antibodies , and red bars denote
P2A -turboGFP, 1E12 of AAV9 - Cas9N -gRNAM3, and 1E12 immunodominant epitopes. AAV9 capsid expresses as three
of AAV9- Cas9N -ORNAMA). M3 or M4, single-site edits; isoforms ( VP1/2 /3 ). (FIG . 8H ) Capsid residues within iden
M3 +M4, double -site edits ; Precise excision , subset of tified epitopes preferentially confer loss of viral blood
M3 +M4 with deletions delimited by the Cas9- gRNAs cut
sites . (FIG . 5F ) AAV9- Cas9- gRNAs preferentially trans persistency when mutated ( Adachi, K., Enoki, T., Kawano ,
duced the liver, heart and skeletal muscle (gastrocnemius Y., Veraz, M. & Nakai, H. Drawing a high -resolution func
and diaphragm ) (*** , P < 0.001; Wilcoxon rank -sum , Bon tional map of adeno - associated virus capsid by massively
ferroni corrected ) (n = 7 , 4E12 ). Red , means = s.e.m .; black parallel sequencing. Nat Commun 5 , 3075 , doi: 10.1038 /
dashed line with gray box , qPCR false positive rate (2.5 ncomms4075 (2014 )), suggesting their association with
vg/dg ) with s.d. (FIG . 5G ) Transduction efficiency with maintaining blood persistency . Each dot represents a double
5E11 of AAV9- Cas9 - gRNAs (**, P < 0.01; *** , P < 0.001; alanine mutated AAVO capsid variant, plotted according to
Wilcoxon rank -sum , Bonferroni corrected ) (n = 9 ). (FIG . 5H ) its measured blood persistency and antigenicity of the resi
Correlation of gene-targeting rates with vg /dg is maintained due . Red bar, mean . (FIG . 81) Capsid residues within iden
at lower dosage (n = 2, 5E11 of AAV9-Cas9- gRNAsM3+M4). tified epitopes preferentially de- targets the liver when
Data from mice injected with 4E12 vg of AAV9-Cas9 mutated (Adachi, K., Enoki, T., Kawano , Y., Veraz , M. &
gRNAsM3 „M3+M4, as shown in FIG . 4B , is reproduced here for Nakai , H. Drawing a high -resolution functional map of
comparison . adeno -associated virus capsid by massively parallel
[0031] FIG . 6 depicts whole-mount epifluorescence sequencing. Nat Commun 5 , 3075 , doi: 10.1038 /
images from neonatal Ai9 mice injected systemically with ncomms4075 ( 2014 )), suggesting their association with
AAV9 -Cas9- gRNAs (5E11 vg ) targeting the 3xStop cassette hepatotropism . Each dot represents a double -alanine
and controls. Numerous tdTomato + cells were observed in mutated AAVO capsid variant, plotted according to its mea
mice injected with AAV9- Cas9 - gRNAs targeting the sured tropism and antigenicity of the residue. Blue bar, mean
genomic 3xStop cassette, but not in negative control liver transduction efficiency; Magenta bar, mean global
vehicle - injected mice, indicating that fluorescence activa transduction efficiency, excluding the liver . Antigenicity ,
tion resulted from 3xStop excision . TdTomato + cells were ranging from 0 to 8 , denotes number of animals in which a
also observed , at lower frequencies, in mice injected with particular residue is part of a linear epitope .
AAV9s encoding two gRNAsboth targeting one side of the [0034 ] FIGS. 9A -D depict additional data for epitope
3xStop cassette (AAV9-Cas9 - gRNAs"Td5 + TdL or AAVE mapping and recoding of AAV9-CRISPR - Cas9. (FIG . 9A )
Cas9-gRNAs Td3+ TdR), suggesting the introduction of large Mapped epitopes for monoclonal (mAb ) and polyclonal
deletions that removed the 3xStop terminators . (pAb) Cas9 - specific antibodies titrated at 200 , 20 , and 2 ug
Gray = dTomato . ml- 1. P -values from Wald test , Benjamini-Hochberg
[0032 ] FIGS. 7A - C depict that tissue sections from Ai9 adjusted for FDR = 0.1 . (FIG . 9B ) Known functional variants
mice injected with AAV9- Cas9 -gRNAs. AAV9-Cas9 - gR of Cas9 ( Jinek , M. et al. Structures of Cas9 endonucleases
NASTAL + TdR (4E12 vg ) transduced multiple organs, excising reveal RNA -mediated conformational activation . Science
the 3xStop genomic locus , as indicated by tdTomato acti 343 , 1247997 , doi: 10.1126 /science.1247997 (2014 ); Klein
vation in (FIG . 7A ) liver, (FIG . 7B ) heart , and (FIG . 7C ) stiver , B. P. et al. Engineered CRISPR - Cas9 nucleases with
skeletal muscle . TdTomato + cells were not detected in altered PAM specificities . Nature 523, 481-485 , doi: 10 .
control mice injected with AAV9- Cas9-gRNAs M3 +M4 Scale 1038 /nature14592 ( 2015 )) can be combined to recode iden
bars, 500 um . tified epitopes. Recoded Cas9 retains endonucleolytic func
[0033] FIGS. 8A -I depict that AAV9 and Cas9 activate tion . Ai9 fibroblasts were lipofected with wild -type or
immune responses . (FIG . 8A ) Intramuscular Cas9-expres variant Cas9 -encoding plasmids, programmed with the indi
sion via AAV9 -split- Cas9 injection or plasmid - Cas9FL elec cated gRNAs, and genomic excision -dependent tdTomato
troporation . (FIG . 8B ) Intramuscular Cas9 expression fluorescence was assayed 4 days post -transfection . Deletion
induces lymphocyte infiltration in the draining inguinal and of the epitope (A1126-1135 ) abolishes Cas9 activity. Scale
popliteal lymph nodes (n = 4 mice per condition ) (* , P < 0.05 ; bar , 500 um . (FIG . 9C ) AAV9 - specific antibodies were
n -way ANOVA ). Checkmarks denote injected vectors and elicited by two weeks post-injection , as determined by
conditions . (FIG . 8C ) TCR - B CDR3 repertoires converged fluorescent immunoassay (FIAX ). Two groups of mice
after Cas9-exposure, indicating Cas9- induced expansion of injected with 4E12 vg AAV9 - Cas9 - VPR -gRNAs are shown,
T-cell subsets (n = 4; 6 pair -wise comparisons) (Welch's differing only in the gRNA spacers employed (** , P < 0.01 ;
t- test, Bonferroni corrected ). (FIG . 8D ) VB16 CDR3 one -way ANOVA , followed by Dunnett's test against
CASSLDRGQDTQYF is a public Cas9 -responsive T-cell vehicle-injected mice ). (FIG . 9D ) AAV9 capsid -specific
clonotype (Welch's t-test). Numbers in parentheses denote epitopes reside predominantly on the capsid surface. Red
clonotypic rank within each TCR -B CDR3 repertoire after bar,mean . Antigenicity , ranging from 0 to 8 , denotes number
Cas9 re-stimulation . (FIG . 8E ) Epitope mapping by M13 of animals in which a particular residue is part of a linear
phage display (all Ig subclasses ). ( FIG . 8F ) Cas9 epitopes epitope .
US 2019/0345483 A1 Nov. 14 , 2019
7
[0035 ] FIGS. 10A - D depict that AAV -CRISPR - Cas9 does [0038 ] In some embodiments, the first nucleic acid esters
not induce effector cytolysis seen with DNA electroporation . a first portion of the Cas9 protein having a first split-mtein
(FIG . 10A ) AAV9-Cas9 -VPR - gRNAs treatment does not and wherein the second nucleic acid encodes a second
elicit intramuscular IL -2 secretion or perforin release (n = 3 portion of the Cas9 protein having a second split - intein
mice per condition ). Mice were targeted with 7 gRNAs complementarity to the first split-intein and wherein the first
against Mstn , Fst, PD -L1 and CD47 (gRNAs set 1) or with portion of the Cas9 protein and the second portion of the
3 gRNAs against Mstn and Fst (gRNAs set 2 ), all at 4E12 Cas9 protein are joined together to form the Cas9 protein . In
vg total of 1:1 AAV9- Cas9N -gRNAs:AAV - Cas9C - VPR . All other embodiments, the first nucleic acid encodes first
injections included 1E11 vg of AAV9- turboRFP to demar portion of the Cas9 protein having a N - split - intein RmaIntN
cate transduction. (FIG . 10B ) IL -2 and perforin protein and wherein the second nucleic acid encodes a second
levels were elevated in muscles electroporated with Cas9 portion of the Cas9 protein having a C -split - intein RmaIntC
encoding DNA (n = 3 mice per condition ). (FIG . 10C ) AAVI and wherein the first portion of the Cas9 protein and the
Cas9 -VPR - gRNAs did not induce detectable myofiber dam second portion of the Cas9 protein are joined together to
age , as assessed by quantifying the fraction of centrally form the Cas9 protein . In certain embodiments, the first
nucleated myofibers (n = 3 mice per condition ) (one-way portion of the Cas9 protein is the N -terminal lobe of the
ANOVA , followed by Dunnett’s test against mice injected Cas9 protein and the second portion of the Cas9 protein is
with 1E11 vg of AAV9- turboRFP ). (FIG . 10D ) Cellular the C -terminal lobe of the Cas9 protein . In certain embodi
damage that causes myofiber degeneration and repair typi ments , the first portion of the Cas9 protein is the N -terminal
cally results in centrally nucleated myofibers under histo lobe of the Cas9 protein up to amino acid 1713 and the
logical examination . Part of a histological section is shown second portion of the Cas9 protein is the C -terminal lobe of
to depict the quantification method . Delivery ofminicircle the Cas9 protein beginning at D714 .
Cas9FL or PCAG -GFP via DNA electroporation induced an [0039 ] Embodiments of the present disclosure are directed
increase in the fraction of centrally nucleated myofibers, to a method of altering a target nucleic acid in a cell of a
compared to controls electroporated with vehicle only. subject . In one embodiment, the method includes delivering
FK506 reduced but did not fully mitigate the elevated to the cell of the subject a first nucleic acid encoding a first
fraction of centrally nucleated myofibers (n = 4 mice per portion of a Cas9 protein and a guide RNA ( GRNA ) wherein
condition ) (* , P < 0.05 ; ** , P < 0.01; *** , P < 0.001; one -way the first nucleic acid is within a first vector, delivering to the
ANOVA , followed by Tukey -Kramer test). cell of the subject a second nucleic acid encoding a second
portion of the Cas9 protein and optionally a transcriptional
DETAILED DESCRIPTION regulator wherein the second nucleic acid is within a second
vector, wherein the cell expresses the first portion of the
[0036 ] Embodiments of the present disclosure are directed Cas9 protein , the gRNA and the second portion of the Cas9
to a method of altering a target nucleic acid in a cell . In one protein or the second portion of the Cas9 and the transcrip
embodiment, themethod includes providing to the cell a first tional regulator fusion protein , wherein the first portion of
nucleic acid encoding a first portion of a Cas9 protein and a the Cas9 protein and the second portion of the Cas9 protein ,
guide RNA (GRNA ), providing to the cell a second nucleic or the first portion of the Cas9 protein and the second portion
acid encoding a second portion of the Cas9 protein and of the Cas9 and the transcriptional regulator fusion protein
optionally a transcriptional regulator, wherein the cell are joined together to form the Cas9 protein or the Cas9
expresses the first portion of the Cas9 protein , the gRNA and fusion protein , and wherein the gRNA and the Cas9 protein ,
the second portion of the Cas9 protein or the second portion or the gRNA and the Cas9 fusion protein form a co
of the Cas9 and the transcriptional regulator fusion protein , localization complex with the target nucleic acid and alter
wherein the first portion of the Cas9 protein and the second the expression of the target nucleic acid . In certain embodi
portion of the Cas9 protein , or the first portion of the Cas9 ment, the Cas9 protein is enzymatically active and the
protein and the second portion of the Cas9 and the tran enzymatically active Cas9 protein cleaves the target nucleic
scriptional regulator fusion protein are joined together to acid in a site specific manner. In some embodiments, the
form the Cas9 protein or the Cas9 fusion protein , and gRNA can have full length or truncated spacer sequence. In
wherein the gRNA and the Cas9 protein , or the gRNA and certain embodiments, the gRNA having a truncated spacer
the Cas9 fusion protein form a co - localization complex with sequence guides the Cas9 protein or the Cas9 fusion protein
the target nucleic acid and alter the expression of the target to the target nucleic acid and regulate the expression of the
nucleic acid . In some embodiments, the Cas9 protein is target nucleic acid without cleaving the target nucleic acid .
enzymatically active and the enzymatically active Cas9 [0040 ] In one embodiment, the first vector is a plasmid or
protein cleaves the target nucleic acid in a site specific adeno- associated virus. In another embodiment, the second
manner. In other embodiments , the gRNA can have full vector is a plasmid or adeno -associated virus.
length or truncated spacer sequence . In certain embodi [0041 ] In some embodiments, the first nucleic acid
ments , the gRNA having a truncated spacer sequence guides encodes a first portion of the Cas9 protein having first
the Cas9 protein or the Cas9 fusion protein to the target split- intein and wherein the second nucleic acid encodes a
nucleic acid and regulate the expression of the target nucleic second portion of the Cas9 protein basing a second split
acid without cleaving the target nucleic acid . intein complementary to the first split- intein and wherein the
[0037 ] In some embodiments , the firstnucleic acid and the first portion ofdie Cas9 protein and the second portion of the
second nucleic acid are delivered to the cell by separate Cas9 protein are joined together to form the Cas9 protein . In
vectors. In one embodiment, the first nucleic acid is deliv other embodiments , the first nucleic acid encodes a first
ered to the cell by a plasmid or adeno -associated virus. In portion of the Cas9 protein having a N - split-intein RmaIntN
another embodiment, the second nucleic acid is delivered to and wherein the second nucleic acid encodes a second
the cell by a plasmid or an adeno -associated virus . portion of the Cas9 protein having a C - split -intein RmaIntC
US 2019/0345483 A1 Nov. 14 , 2019
8
and wherein the first portion of the Cas9 protein and the [0048 ] The cell according to some embodiments of the
second portion of the Cas9 protein are joined together to present disclosure is a eukaryotic cell or a prokaryotic cell .
form die Cas9 protein . In one embodiment, the first portion In certain embodiments, the cell is a bacteria cell , a yeast
of the Cas9 protein is the N -terminal lobe of the Cas9 protein cell, a mammalian cell, a human cell , a plant cell or an
and the second portion of die Cas9 protein is the C -terminal animal cell . In some embodiments, the cell is in vitro , in vivo
lobe of the Cas9 protein . In another embodiment, the first or ex vivo .
portion of the Cas9 protein is the N - terminal lobe of die [0049 ] The split Cas9 methods described herein are useful
Cas9 protein up to amino acid V713 and the second portion in CRISPR -related methods where Cas9 and a guide RNA
of the Cas9 protein is the C -terminal lobe of the Cas9 protein are used to colocalize the Cas9 and the guide RNA to a target
beginning at D714 . nucleic acid sequence. Accordingly, embodiments of the
[0042 ] In some embodiments , the vectors are delivered to present disclosure are based on the use ofRNA guided DNA
the cell of the subject via various routes known to a skilled binding proteins, such as Cas9 , to co -localize with guide
in the art including systemic , local , intravenous, intraperi RNA at a target DNA site . Such DNA binding proteins are
toneal, intramuscular routes or via injection or electropora readily known to those of skill in the art to bind to DNA for
tion . The subject of the disclosure includes human , patients various purposes . Such DNA binding proteinsmay be natu
or an animal. No overt cellular or tissue damage is observed rally occurring . DNA binding proteins included within the
when the vectors are adeno - associated viruses . scope of the present disclosure include those which may be
[0043 ] Embodiments of the present disclosure are directed guided by RNA , referred to herein as guide RNA . According
to a method ofmodulating a target gene expression in a cell. to one aspect, the guide RNA is between about 10 to about
In one embodiment , the method includes providing to the 500 nucleotides. According to one aspect, the RNA is
cell a first recombinant adeno-associated virus comprising a between about 20 to about 100 nucleotides. According to
first nucleic acid encoding an N -terminal portion of the Cas9 this aspect, the guide RNA and the RNA guided DNA
protein (Cas9M) and a gRNA, providing to the cell a second binding protein form a co - localization complex at the DNA .
recombinant adeno -associated virus comprising a second [0050 ] DNA binding proteins having nuclease activity are
nucleic acid encoding a fusion protein comprising a C -ter known to those of skill in the art , and include naturally
minal portion of the Cas9 protein ( Cas99) fused with a occurring DNA binding proteins having nuclease activity ,
transcriptional regulator ( TR ), wherein the cell expresses the such as Cas9 proteins present, for example, in Type II
Cas9N protein and the Cas9C - TR fusion protein and join CRISPR systems. Such Cas9 proteins and Type II CRISPR
them to form a full length Cas9FL - TR fusion protein , and systems are well documented in the art. See Makarova et al.,
wherein the cell expresses the gRNA and the gRNA directs Nature Reviews, Microbiology, Vol. 9, June 2011, pp . 467
the Cas9FL- TR fusion protein to the target gene and modu 477 including all supplementary information hereby incor
lates target gene expression . porated by reference in its entirety . Exemplary Cas include
[0044 ] In some embodiments , the transcriptional regulator S. pyogenes Cas9 ( SpCas9 ), S. aureus Cas9 (SaCas9 ) and S.
is a transcriptional activator or a transcriptional repressor. In thermophilus Cas9 (StCas9). Additional exemplary CRISPR
one embodiment, the transcriptional activator is VPR . In systems include Cpfl proteins for RNA -guided genome
certain embodiments , the transcriptional regulator is a editing . See Zetsche , B. et al. Cpf1 is a single RNA - guided
recruiter protein that recruits epigenetic modulators to the endonuclease of a class 2 CRISPR -Cas system . Cell, 2015 ,
target gene . In exemplary embodiments, the gRNA has 163, 759-771. Additional exemplary nucleic -acid guided
truncated spacer sequence and directs Cas9FL - TR fusion systems include argonaute proteins for DNA -guided
protein binding to target DNA without cleaving the target genome-editing. See Gao F, Shen X Z , Jiang F , Wu Y , Han
DNA . C , DNA - guided genome editing using the Natronobacte
[0045 ] Embodiments of the present event disclosure are rium gregoryi Argonaute, Nat Biotechnol., 2016 May 2. doi:
directed to a method of imaging a target nucleic acid in a 10.1038 /nbt.3547 hereby incorporated by reference in its
entirety .
cell. In one embodiment, the method includes providing to [0051 ] Bacterial and archaeal CRISPR -Cas systems rely
the cell a first recombinant adeno -associated virus compris on short guide RNAs in complex with Cas proteins to direct
ing a first nucleic acid encoding an N - terminal portion of the degradation of complementary sequences present within
Cas9 protein (Cas9M ) and a gRNA , providing to the cell a invading foreign nucleic acid . See Deltcheva , E. et al.
second recombinant adeno -associated virus comprising a CRISPR RNA maturation by trans - encoded small RNA and
second nucleic acid encoding a fusion protein comprising a host factor RNase III. Nature 471, 602-607 (2011 ); Gasiu
C -terminal portion of the Cas9N protein (Cas99 fused with nas, G., Barrangou , R., Horvath , P. & Siksnys, V. Cas9
a fluorescein protein , wherein the cell expresses the Cas9N CrRNA ribonucleoprotein complex mediates specific DNA
protein and the Cas9 ° fluorescent fusion protein and join cleavage for adaptive immunity in bacteria. Proceedings of
them to form a full length Cas9FL fluorescent fusion protein , the National Academy of Sciences of the United States of
and wherein the cell expresses the gRNA , and the gRNA America 109, E2579-2586 ( 2012); Jinek , M. et al. A pro
directs the Cas9FL fluorescein fusion protein to the target grammable dual- RNA - guided DNA endonuclease in adap
nucleic acid and produces fluorescent imagine of the target
nucleic acid . tive bacterial immunity . Science 337 , 816-821 (2012 ); Sap
ranauskas, R. et al. The Streptococcus thermophilus
[0046 ] In an exemplary embodiment, the Cas9 is a Type II CRISPR /Cas system provides immunity in Escherichia coli .
CRISPR system Cas9. In some embodiments, the Cas9 Nucleic acids research 39, 9275-9282 (2011) ; and Bhaya ,
protein is an enzymatically active Cas9 protein , a Cas9 D., Davison , M. & Barrangou , R. CRISPR - Cas systems in
protein nickase or a nuclease null Cas9 protein . bacteria and archaea: versatile small RNAs for adaptive
[0047 ] In some embodiments, the Cas9N protein and the defense and regulation . Annual review of genetics 45 , 273
Cas9C - TR fusion protein are joined by split inteins. 297 (2011). A recent in vitro reconstitution of the S. pyo
US 2019/0345483 A1 Nov. 14 , 2019
9
genes type II CRISPR system demonstrated that crRNA (January, 2011 ); Kleinstiver B P ,Prew MS, Tsai SQ , Topkar
(“ CRISPR RNA ” ) fused to a normally trans- encoded tracr VV, Nguyen N T, Zheng Z , Gonzales A P , Li Z , Peterson R
RNA (“ trans - activating CRISPR RNA ” ) is sufficient to T, Yeh JR , Aryee M J, Joung J K , Engineered CRISPR - Cas9
direct Cas9 protein to sequence -specifically cleave target nucleases with altered PAM specificities, Nature. 2015 Jul.
DNA sequences matching the crRNA . Expressing a gRNA 23 ; 523 (7561): 481-5 ; Kleinstiver B P , Prew MS, Tsai SQ,
complementary to a target site results in Cas9 recruitment Nguyen N T , Topkar V V , Zheng Z , Joung J K , Broadening
and degradation of the target DNA . See H. Deveau et al., the targeting range of Staphylococcus aureus CRISPR -Cas9
Phage response to CRISPR -encoded resistance in Strepto by modifying PAM recognition , Nat Biotechnol. 2015
coccus thermophilus. Journal of Bacteriology 190 , 1390 December ; 33 ( 12 ): 1293-1298 , each of which are hereby
(February, 2008). incorporated by reference in their entireties . According to
[0052 ] Two classes of CRISPR systems are generally one aspect, a particular useful enzyme according to the
known and are referred to as class 1 and class 2. Class 1 present disclosure to cleave dsDNA is the single effector
systems can be further classified into types I, III, and IV , enzyme, Cpf1, belonging to Type V. See Zetsche , B. et al.
while class 2 systems can be further classified into type II Cpfi is a single RNA- guided endonuclease of a class 2
and V. According to one aspect, a particular useful enzyme CRISPR -Cas system . Cell, 2015 , 163, 759-771 .
according to the present disclosure to cleave dsDNA is the [0054 ] Exemplary DNA binding proteins having nuclease
single effector enzyme, Cas9, common to Type II. See K. S. activity function to nick or cut double stranded DNA. Such
Makarova et al., Evolution and classification of the nuclease activity may result from the DNA binding protein
CRISPR -Cas systems. Nature reviews. Microbiology 9 , 467 having one or more polypeptide sequences exhibiting nucle
( June, 2011) hereby incorporated by reference in its entirety . ase activity . Such exemplary DNA binding proteins may
Within bacteria, the Type II effector system consists of a have two separate nuclease domains with each domain
long pre -crRNA transcribed from the spacer -containing responsible for cutting or nicking a particular strand of the
CRISPR locus, the multifunctional Cas9 protein , and a double stranded DNA. Exemplary polypeptide sequences
tracrRNA important for gRNA processing . The tracrRNAS having nuclease activity known to those of skill in the art
hybridize to the repeat regions separating the spacers of the include the McrA -HNH nuclease related domain and the
pre -crRNA , initiating dsRNA cleavage by endogenous RuvC - like nuclease domain . Accordingly, exemplary DNA
RNase III, which is followed by a second cleavage event binding proteins are those that in nature contain one or more
within each spacer by Cas9, producing mature crRNAs that of the MerA - HNH nuclease related domain and the RuvC
remain associated with the tracrRNA and Cas9 . TracrRNA like nuclease domain .
crRNA fusions are contemplated for use in the present [0055 ] In S. pyogenes , Cas9 generates a blunt-ended
methods. double -stranded break 2-4 bp upstream of the protospacer
[0053 ] According to one aspect, the enzymeof the present adjacent motif (PAM ) via a process mediated by two cata
disclosure, such as Cas9 unwinds the DNA duplex and lytic domains in the protein : an HNH domain that cleaves
searches for sequences matching the crRNA to cleave . the complementary strand of the DNA and a RuvC - like
Target recognition occurs upon detection of complementar domain that cleaves the non -complementary strand . See
ity between a “ protospacer” sequence in the target DNA and Jinek et al., Science 337 , 816-821 (2012 ) hereby incorpo
the remaining spacer sequence in the crRNA . Importantly , rated by reference in its entirety . Cas9 proteins are known to
Cas9 cuts the DNA only if a correct protospacer-adjacent exist in many Type II CRISPR systems including the fol
motif (PAM ) is also present at the 3' end . According to lowing as identified in the supplementary information to
certain aspects, different protospacer- adjacent motif can be Makarova et al.,Nature Reviews, Microbiology, Vol. 9, June
utilized . For example , the S. pyogenes system requires an 2011, pp . 467-477 : Methanococcus maripaludis C7;
NGG sequence , where N can be any nucleotide. S. thermo Corynebacterium diphtheriae; Corynebacterium efficiens
philus Type II systemsrequire NGGNG (see P. Horvath , R. YS -314 ; Corynebacterium glutamicum ATCC 13032
Barrangou , CRISPR /Cas, the immune system of bacteria Kitasato ; Corynebacterium glutamicum ATCC 13032
Bielefeld ; Corynebacterium glutamicum R ; Corynebacte
and archaea . Science 327 , 167 (Jan. 8 , 2010 ) hereby incor
porated by reference in its entirety and NNAGAAW (see H. rium kroppenstedtii DSM 44385; Mycobacterium abscessus
Deveau et al., Phage response to CRISPR -encoded resis ATCC 19977 ; Nocardia farcinica IFM10152 ; Rhodococcus
tance in Streptococcus thermophilus. Journal of bacteriology erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus
190 , 1390 (February, 2008 ) hereby incorporated by refer opacus B4 uid36573 ; Acidothermus cellulolyticus 11B ;
ence in its entirety ), respectively, while different S. mutans Arthrobacter chlorophenolicus A6; Kribbella flavida DSM
systems tolerate NGG or NAAR (see J. R. van der Ploeg , 17836 uid43465; Thermomonospora curvata DSM 43183;
Analysis of CRISPR in Streptococcus mutans suggests fre Bifidobacterium dentium Bdi ; Bifidobacterium longum
quent occurrence of acquired immunity against infection by DJO10A ; Slackia heliotrinireducens DSM 20476 ; Perse
M102 -like bacteriophages. Microbiology 155, 1966 (June, phonella marina EX H1; Bacteroides fragilis NCTC 9434 ;
2009 ) hereby incorporated by reference in its entirety . Bio Capnocytophaga ochracea DSM 7271 ; Flavobacterium
informatic analyses have generated extensive databases of psychrophilum JIPO2 86 ; Akkermansia muciniphila ATCC
CRISPR loci in a variety of bacteria that may serve to BAA 835 ; Roseiflexus castenholzii DSM 13941; Roseiflexus
identify additional useful PAMs and expand the set of RS1; Synechocystis PCC6803 ; Elusimicrobium minutum
CRISPR -targetable sequences (see M. Rho, Y. W. Wu, H. Pei191; uncultured Termite group 1 bacterium phylotype Rs
Tang , T. G. Doak , Y. Ye , Diverse CRISPRs evolving in D17 ; Fibrobacter succinogenes S85 ; Bacillus cereus ATCC
human microbiomes . PLoS genetics 8 , e1002441 ( 2012) and 10987; Listeria innocua ; Lactobacillus casei; Lactobacillus
D. T. Pride et al., Analysis of streptococcal CRISPRs from rhamnosus GG ; Lactobacillus salivarius UCC118 ; Strepto
human saliva reveals substantial sequence diversity within COCCUS agalactiae A909 ; Streptococcus agalactiae
and between subjects over time. Genome research 21 , 126 NEM316 ; Streptococcus agalactiae 2603 ; Streptococcus
US 2019/0345483 A1 Nov. 14 , 2019
10
dysgalactiae equisimilis GGS 124 ; Streptococcus equi - continued

zooepidemicus MGCS10565 ; Streptococcus gallolyticus NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD
UCN34 uid46061; Streptococcus gordonii Challis subst LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
CH1; Streptococcus mutansNN2025 uid46353; Streptococ
cus mutans, Streptococcus pyogenes M1 GAS ; Streptococ IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKO
CUS pyogenes MGAS5005 ; Streptococcus pyogenes LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD
MGAS2096 ; Streptococcus pyogenes MGAS9429 ; Strepto
coccus pyogenes MGAS10270 ; Streptococcus pyogenes SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV
MGAS6180 ; Streptococcus pyogenes MGAS315 ; Strepto MGRHKPENIVIEMARENOTTOKGOKNSRERMKRIEEGIKELGSQILKEHP
COCCUS pyogenes SSI- 1 ; Streptococcus pyogenes
MGAS10750 ; Streptococcus pyogenes NZ131; Streptococ VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
cus thermophiles CNRZ1066 , Streptococcus thermophiles SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWROLLNAKLITORKFDNL
LMD - 9; Streptococcus thermophiles LMG 18311 ;
Clostridium botulinum A3 Loch Maree ; Clostridium botu TKAERGGLSELDKAGFIKROLVETRQITKHVAQILDSRMNTKYDENDKLI
linum B Eklund 17B ; Clostridium botulinum Ba4 657; REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
Clostridium botulinum F Langeland; Clostridium cellulolyti
cum H10 ; Finegoldia magna ATCC 29328 ; Eubacterium YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKIEI
rectale ATCC 33656 ; Mycoplasma gallisepticum ; Myco
plasma mobile 163K ; Mycoplasma penetrans; Mycoplasma TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
synoviae 53 ; Streptobacillus moniliformis DSM 12112 ; Bra QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE
dyrhizobium BTAil ; Nitrobacter hamburgensis X14 ; Rho
dopseudomonas palustris BisB18; Rhodopseudomonas KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
palustris BisB5; Parvibaculum lavamentivorans DS- 1 ; YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPE
Dinoroseobacter shibae DFL 12 ; Gluconacetobacter diazo
trophicus Pal 5 FAPERJ; Gluconacetobacter diazotrophicus DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
Pal 5 JGI; Azospirillum B510 uid46085 ; Rhodospirillum
rubrum ATCC 11170 ; Diaphorobacter TPSY uid29975 ; PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
Verminephrobacter eiseniae EF01-2 ; Neisseria meningitides SITGLYETRIDLSQLGGD
053442 ; Neisseria meningitides alpha14 ; Neisseria menin
gitides Z2491 ; Desulfovibrio salexigens DSM 2638 ; [0056 ] According to one aspect , the specificity of gRNA
Campylobacter jejuni doylei 269 97 ; Campylobacter jejuni directed Cas9 cleavage is used as a mechanism for genome
81116 ; Campylobacter jejuni; Campylobacter lari RM2100; engineering . According to one aspect, hybridization of the
Helicobacter hepaticus; Wolinella succinogenes; Tolumonas ORNA need not be 100 percent in order for the enzyme to
auensis DSM 9187; Pseudoalteromonas atlantica T6c ; recognize the gRNA /DNA hybrid and affect cleavage. Some
Shewanella pealeana ATCC 700345 ; Legionella pneumo off-target activity could occur. For example , the S. pyogenes
phila Paris ; Actinobacillus succinogenes 130Z ; Pasteurella system tolerates mismatches in the first 6 bases out of the 20
multocida ; Francisella tularensis novicida U112 ; Franci bp mature spacer sequence in vitro . According to one aspect,
sella tularensis holarctica ; Francisella tularensis FSC 198 ; greater stringency may be beneficial in vivo when potential
Francisella tularensis tularensis; Francisella tularensis off- target sites matching (last 14 bp ) NGG do not exist
WY96-3418 ; and Treponema denticola ATCC 35405. The within the human reference genome for the gRNAs .
Cas9 protein may be referred by one of skill in the art in the
literature as Csn1. An exemplary S. pyogenes Cas9 protein [0057 ] According to certain aspects, specificity may be
sequence is shown below . See Deltcheva et al., Nature 471, improved . When interference is sensitive to the melting
602-607 (2011) hereby incorporated by reference in its temperature of the gRNA -DNA hybrid , AT-rich target
entirety . sequences may have fewer off -target sites. Carefully choos
ing target sites to avoid pseudo -sites with at least 14 bp
matching sequences elsewhere in the genome may improve
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA specificity . According to certain aspect , the gRNAs can be
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR designed to include 16-18 nucleotide spacers, which
increases specificity while retaining Cas9 endonucleolytic
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS TDKAD activity (Fu Y , Sander J D , Reyon D , Cascio V M , Joung J
LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
K , Improving CRISPR -Cas nuclease specificity using trun
cated guide RNAs, Nat Biotechnol., 2014 March ; 32( 3 ):279
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 84, hereby incorporated by reference in its entirety ). The use
of a Cas9 variant requiring a longer PAM sequence may
NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI reduce the frequency of off - target sites . Directed evolution
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI may improve Cas9 specificity to a level sufficient to com
pletely preclude off -target activity , ideally requiring a per
FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR fect 20 bp gRNA match with a minimal PAM . Accordingly ,
KORTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY modification to the Cas9 protein is a representative embodi
ment of the present disclosure . CRISPR systems useful in
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK the present disclosure are described in R. Barrangou , P.
Horvath , CRISPR : new horizons in phage resistance and
strain identification . Annual review of food science and
US 2019/0345483 A1 Nov. 14 , 2019
11
technology 3 , 143 ( 2012 ) and B. Wiedenheft, S. H. Stern CRISPR System which lacks nuclease activity . An exem
berg, J. A. Doudna, RNA -guided genetic silencing systems plary DNA binding protein is a nuclease -null Cpf1 protein .
in bacteria and archaea . Nature 482, 331 (Feb. 16 , 2012 ) [0062 ] According to one aspect, the Cas9 protein , Cas9
each ofwhich are hereby incorporated by reference in their protein nickase or nuclease null Cas9 includes homologs and
entireties. orthologs thereof which retain the ability of the protein to
[0058 ] Guide RNAs useful in the disclosed methods bind to the DNA and be guided by the RNA . According to
include those having a spacer sequence , a tracr mate one aspect, the Cas9 protein includes the sequence as set
sequence and a tracr sequence, with the spacer sequence forth for naturally occurring Cas9 from S. pyogenes and
being between about 16 to about 20 nucleotides in length protein sequences having at least 30 % , 40 % , 50 % , 60 % ,
and with the tracr sequence being between about 60 to about 70 % , 80 % , 90 % , 95 % , 98 % or 99 % homology thereto and
500 nucleotides in length and with a portion of the tracr being a DNA binding protein , such as an RNA guided DNA
sequence being hybridized to the tracr mate sequence and binding protein .
with the tracr mate sequence and the tracr sequence being [0063] According to one aspect, an engineered Cas9
linked by a linker nucleic acid sequence of between about 4 ORNA system is provided wherein one or more of functional
to about 6 nucleotides. crRNA -tracrRNA fusions are con groups or function -conferring domains such as Foki het
templated as exemplary guide RNA. erodimers (see Tsai, S. Q., et al., Dimeric CRISPR RNA
[0059 ] According to certain aspects, the DNA binding guided Fokl nucleases for highly specific genome editing .
protein is altered or otherwise modified to inactivate the Nat Biotechnol, 2014. 32 (6 ): p . 569-76 , and Guilinger, J. P.,
nuclease activity . Such alteration or modification includes D. B. Thompson , and D. R. Liu , Fusion of catalytically
altering one or more amino acids to inactivate the nuclease inactive Cas9 to Foki nuclease improves the specificity of
activity or the nuclease domain . Such modification includes genomemodification . Nat Biotechnol, 2014. 32 (6 ): p . 577
removing the polypeptide sequence or polypeptide 82. ), transcriptional regulators (see Gilbert , L. A., et al.,
sequences exhibiting nuclease activity, i.e. the nuclease CRISPR -mediated modular RNA -guided regulation of tran
domain , such that the polypeptide sequence or polypeptide scription in eukaryotes. Cell, 2013. 154 (2 ): p . 442-51 ,Mali ,
sequences exhibiting nuclease activity , i.e.nuclease domain , P., et al., CAS9 transcriptional activators for target specific
are absent from the DNA binding protein . Other modifica ity screening and paired nickases for cooperative genome
tions to inactivate nuclease activity will be readily apparent engineering . Nat Biotechnol, 2013. 31 (9 ): p . 833-8 , Perez
to one of skill in the art based on the present disclosure . Pinera , P., et al., RNA - guided gene activation by CRISPR
Accordingly , a nuclease -null DNA binding protein includes Cas9 -based transcription factors . Nat Methods, 2013 .
polypeptide sequences modified to inactivate nuclease activ 10 ( 10 ): p . 973-6 , and Cheng , A. W., et al., Multiplexed
ity or removal of a polypeptide sequence or sequences to activation of endogenous genes by CRISPR - on , an RNA
inactivate nuclease activity. The nuclease-null DNA binding guided transcriptional activator system . Cell Res , 2013.
protein retains the ability to bind to DNA even though the 23 ( 10 ): p . 1163-71 ), fluorescent proteins (see Gilbert , L. A.,
nuclease activity has been inactivated . Accordingly , the et al ., CRISPR -mediated modular RNA -guided regulation of
DNA binding protein includes the polypeptide sequence or transcription in eukaryotes. Cell, 2013. 154 (2): p. 442-51
sequences required for DNA binding but may lack the one and Chen , B., et al., Dynamic imaging of genomic loci in
ormore or all of the nuclease sequences exhibiting nuclease living human cells by an optimized CRISPR /Cas system .
activity . Accordingly, the DNA binding protein includes the Cell, 2013. 155( 7) : p . 1479-91), protein -protein interacting
polypeptide sequence or sequences required for DNA bind domains (see Tanenbaum , M. E., et al., A protein - tagging
ing but may have one or more or all of the nuclease system for signal amplification in gene expression and
sequences exhibiting nuclease activity inactivated . fluorescence imaging . Cell, 2014. 159 ( 3): p . 635-46 ),
nucleotide base editors (see Komor, A. C., Kim , Y. B.,
[0060 ] According to one aspect, a DNA binding protein Packer , M. S., Zuris, J. A. & Liu , D. R. Programmable
having two or more nuclease domains may be modified or editing of a target base in genomic DNA without double
altered to inactivate all but one of the nuclease domains. stranded DNA cleavage . Nature , doi: 10.1038 /nature17946
Such a modified or altered DNA binding protein is referred (2016 )), and degradation tags are attached to either the Cas9
to as a DNA binding protein nickase , to the extent that the protein or the gRNA or both for delivery to a target nucleic
DNA binding protein cuts or nicks only one strand ofdouble acid .
stranded DNA . When guided by RNA to DNA , the DNA [0064 ] According to one aspect , the transcriptional regu
binding protein nickase is referred to as an RNA guided lator protein or domain is a transcriptional activator. In an
DNA binding protein nickase . exemplary embodiment, the transcriptional activator is
[0061] An exemplary DNA binding protein is an RNA VPR . According to one aspect , the transcriptional regulator
guided DNA binding protein nuclease of a Type II CRISPR protein or domain upregulates expression of the target
System , such as a Cas9 protein or modified Cas9 or homolog nucleic acid. According to one aspect, the transcriptional
of Cas9 or ortholog of Cas9 . An exemplary DNA binding regulator protein or domain is a transcriptional repressor.
protein is an RNA guided DNA binding protein nuclease of According to one aspect , the transcriptional regulator pro
a Type V CRISPR System , such as a Cpfl protein or tein or domain downregulates expression of the target
modified Cpfl or homolog of Cpfl or ortholog of Cpfl. An nucleic acid . Transcriptional activators and transcriptional
exemplary DNA binding protein is a Cas9 protein nickase . repressors can be readily identified by one of skill in the art
An exemplary DNA binding protein is an RNA guided DNA based on the present disclosure .
binding protein of a Type II CRISPR System which lacks [0065 ] According to one aspect, two or more guide RNAs
nuclease activity. An exemplary DNA binding protein is a are provided with each guide RNA being complementary to
nuclease -null Cas9 protein . An exemplary DNA binding an adjacent site in the DNA target nucleic acid . At least one
protein is an RNA guided DNA binding protein of a Type V RNA guided DNA binding protein nickase is provided and
US 2019/0345483 A1 Nov. 14 , 2019
12
being guided by the two or more RNAs, wherein the at least include endogenous ( or naturally occurring ) nucleic acids
one RNA guided DNA binding protein nickase co -localizes and exogenous (or foreign ) nucleic acids . One of skill based
with the two or more RNAs to the DNA target nucleic acid on the present disclosure will readily be able to identify or
and nicks the DNA target nucleic acid resulting in two or design guide RNAs and Cas9 proteins which co -localize to
more adjacent nicks. According to certain aspects, the two or a DNA including a target nucleic acid . One of skill will
more adjacent nicks are on the same strand of the double further be able to identify transcriptional regulator proteins
stranded DNA . According to one aspect, the two or more or domains which likewise co -localize to a DNA including
adjacent nicks are on the same strand of the double stranded a target nucleic acid . DNA includes genomic DNA, mito
DNA and result in homologous recombination . According to chondrial DNA , viral DNA or exogenous DNA . CRISPR
one aspect, the two or more adjacent nicks are on different Cas9 system can also be programmed to target RNA (See ,
strands of the double stranded DNA . According to one O'ConnellMR, Oakes B L , Sternberg SH , East-Seletsky A ,
aspect , the two or more adjacent nicks are on different Kaplan M , Doudna J A , Programmable RNA recognition
strands of the double stranded DNA and create double and cleavage by CRISPR /Cas9, Nature, 2014 , December 11;
stranded breaks. According to one aspect , the two or more 516 (7530 ):263-6 ; Nelles D A , Fang MY, O'Connell M , Xu
adjacent nicks are on different strands of the double stranded J L ,Markmiller S J, Doudna JA , Yeo G W , Programmable
DNA and create double stranded breaks resulting in nonho RNA Tracking in Live Cells with CRISPR /Cas9 , Cell, 2016 ,
mologous end joining . According to one aspect, the two or April 7 : 165(2 ) 488-96 , each of which is hereby incorporated
more adjacent nicks are on different strands of the double by reference in its entirety ) .
stranded DNA and are offset with respect to one another. [0068 ] Foreign nucleic acids ( i.e. those which are not part
According to one aspect, the two or more adjacent nicks are of a cell's natural nucleic acid composition ) may be intro
on different strands of the double stranded DNA and are duced into a cell using any method known to those skilled
offset with respect to one another and create double stranded in the art for such introduction . Such methods include
breaks . According to one aspect, the two or more adjacent transfection , transduction , viral transduction , microinjec
nicks are on different strands of the double stranded DNA tion , electroporation , lipofection , nucleofection , nanopar
and are offset with respect to one another and create double ticle bombardment, transformation, conjugation and the like
stranded breaks resulting in nonhomologous end joining . One of skill in the art will readily understand and adapt such
According to one aspect, the two ormore adjacentnicks are methods using readily identifiable literature sources.
on different strands of the double stranded DNA and create [0069 ] Transcriptional regulator proteins or domains
double stranded breaks resulting in fragmentation of the which are transcriptional activators or transcriptional repres
target nucleic acid thereby preventing expression of the sors may be readily identifiable , by those skilled in the art
target nucleic acid . based on the present disclosure
[0066 ] According to certain aspects, binding specificity of [0070 ] Vectors used to deliver the nucleic acids to cells as
the RNA guided DNA binding protein may be increased described herein include vectors known to those of skill in
according to methods described herein . According to one the art and used for such purposes . Certain exemplary
aspect , off-set nicks are used in methods of genome-editing vectors may be plasmids or adeno -associated viruses known
( see Mali , P. et al. CAS9 transcriptional activators for target to those of skill in the art . AAVs are highly prevalent within
specificity screening and paired nickases for cooperative the human population (see Gao , G., et al., Clades of Adeno
genome engineering . Nature biotechnology, 31, 833-838 , associated viruses are widely disseminated in human tissues
doi:10.1038 /nbt.2675 (2013 ) hereby incorporated by refer J Virol. 2004. 78 ( 12 ): p . 6381-8 , and Boutin . S., et al.,
ence in its entirety ). A large majority of nicks seldom result Prevalence of serum IgG and neutralizing factors against
in NHEJ events, (see Certo et al., Nature Methods 8 , adeno -associated virus (AAV ) types 1, 2,5,6,8 , and 9 in the
671-676 (2011) hereby incorporated by reference in its healthy population , implications forgone therapy using AAV
entirety ) thus minimizing the effects of off- target nicking . In vectors . Hum Gene Ther. 2010. 21 (6 ): p . 704-12 ) and are
contrast, inducing off-set nicks to generate double stranded useful as viral vectors . Many serotypes exist, each with
breaks (DSBs ) is highly effective at inducing gene disrup different tropism for tissue types (see Zincarelli, C., et al.,
tion . According to certain aspects, 5 ' overhangs generate Analysis of AAV serotypes 1-9 mediated gene expression
more significant NHEJ events as opposed to 3' overhangs . and tropism in mice after systemic injection . Mol Ther,
Similarly , 3' overhangs favor HR over NHEJ events , 2008. 16 (6 ): p . 1073-80 ),which allows specific tissues to be
although the total number of HR events is significantly preferentially targeted with appropriate pseudotyping . Some
lower than when a 5' overhang is generated . Accordingly , serotypes, such as serotypes 8 , 9, and rh10 , transduce the
methods are provided for using nicks for homologous mammalian body . See Zincarelli , C., et al. Analysis of AAV
recombination and off-set nicks for generating double serotypes 1-9 mediated gene expression and tropism in mice
stranded breaks to minimize the effects of off- target Cas9 after systemic injection .Mol Ther, 2008. 16 (6 ): p . 1073-80 ,
ORNA activity . Inagaki, K., et al., Robust systemic transduction with AAVI
[0067 ] Target nucleic acids include any nucleic acid vectors in mice : efficient global cardiac gene transfer supe
sequence to which a co - localization complex as described rior to that of AAV8. Mol Ther, 2006. 14 ( 1): p . 45-53 ,
herein can be useful to either cut, nick , regulate or bind . Keeler , A. M., et al., Long - term correction of very long
Target nucleic acids include genes . For purposes of the chain acyl-coA dehydrogenase deficiency in mice using
present disclosure, DNA , such as double stranded DNA , can AAV9 gene therapy .Mol Ther, 2012. 20 (6 ): p . 1131-8 , Gray,
include the target nucleic acid and a co -localization complex S. J., et al., Preclinical differences of intravascular AAVO
can bind to or otherwise co -localize with the DNA at or delivery to neurons and glia : a comparative study of adult
adjacent or near the target nucleic acid and in a manner in mice and nonhuman primates. Mol Ther , 2011. 19 (6 ): p .
which the co - localization complex may have a desired effect 1058-69 , Okada , H., et al., Robust Long-term Transduction
on the target nucleic acid . Such target nucleic acids can of Common Marmoset Neuromuscular Tissue With rAAV1
US 2019/0345483 A1 Nov. 14 , 2019
13
and rAAV9. Mol Ther Nucleic Acids, 2013. 2 : p . e95 , and 1116-21 ), Sp Cas9 has a least restrictive PAM and most
Foust,K.D., et al., Intravascular AAVO preferentially targets consistent efficacy , but its size (4.2 kb ) makes packaging into
neonatal neurons and adult astrocytes. Nat Biotechnol, 2009 . AAV challenging ,necessitating use of a limited repertoire of
27 (1 ): p . 59-65. AAVI has been demonstrated to cross the compact regulatory elements (< 500 bp ) (see Swiech , L., et
blood - brain barrier (see Foust, K. D., et al., Intravascular al., In vivo interrogation of gene function in the mammalian
AAVI preferentially targets neonatal neurons and adult brain using CRISPR -Cas9 . Nat Biotechnol, 2014 and Senis ,
astrocytes . Nat Biotechnol, 2009. 27 ( 1 ): p . 59-65, and E. et al. CRISPR /Cas9-mediated genome engineering : an
Rahim , A. A., et al., Intravenous administration of AAV2/ 9 adeno -associated viral (AAV ) vector toolbox . Biotechnol
to the fetal and neonatal mouse leads to differential targeting ogy journal, 2014 , 9 : p . 1402-1412 ; Long C , Amoasii L ,
of CNS cell types and extensive transduction of the nervous Mireault A A , McAnally J R , Li H , Sanchez -Ortiz E ,
system . FASEB J, 2011. 25 ( 10 ): p . 3505-18 ) that is inac Bhattacharyya S , Shelton J M , Bassel-Duby R , Olson E N ,
cessible to many viral vectors and biologics. Certain AAVs Postnatal genome editing partially restores dystrophin
have a payload of 4.7-5.0 kb (including viral inverted expression in a mouse model of muscular dystrophy, Sci
terminal repeats (ITRs), which are required in cis for viral ence, 2016 January 22 ; 351 (6271 ): 400-3 ; Yang Y, Wang L ,
packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of Bell P , McMenamin D , He Z , White J, Yu H , Xu C ,
genome size on AAV vector packaging . Mol Ther, 2010 . Morizono H , Musunuru K , Batshaw ML, Wilson J M , A
18 ( 1 ): p . 80-6 and Dong , J. Y., P. D.Fan , and R. A. Frizzell , dual AAV system enables the Cas9-mediated correction of a
Quantitative analysis of the packaging capacity of recom metabolic liver disease in newborn mice , Nat Biotechnol.,
binant adeno - associated virus. Hum Gene Ther , 1996. 7 ( 17 ): 2016 , March ; 34 (3 ):334-8; each ofwhich are hereby incor
p . 2101-12. porated by reference in its entirety ), and precluding the
[0071 ] Delivery methods commonly used in research , fusion of function -conferring domains. The Cas9 protein
such as lentiviruses , adenoviruses, or nucleic - acid -com was re -engineered to eliminate this obstacle .
plexes, exhibit substantial immunogenic and cytotoxic prop [0072] According to certain aspects , two or more portions
erties, which can further compound the immunogenicity of a Cas9 protein are provided within a cell or are otherwise
from ectopic transgene -expression . Furthermore, these expressed within a cell and are combined together to form
approaches generally lack the capacity for targeting of the Cas9 protein . This structure - guided design is essential
specific tissues and for robust full -body delivery. To simul since splitting Cas9 at ordered protein regions significantly
taneously minimize pathological impacts and enable sys impacts function . See Nishimasu , H. et al. Crystal structure
temic genome editing, CRISPR was delivered via adeno of Cas9 in complex with guide RNA and target DNA . Cell
associated viruses (AAVs). AAVs are prevalent within 156 , 935-949 (2014 ), Jinek , M. et al. Structures of Cas9
human populations (see Gao , G., et al ., Clades of Adeno endonucleases reveal RNA -mediated conformational acti
associated viruses are widely disseminated in human tissues . vation . Science 343, 1247997 (2014 ), Zetsche, B., Volz , S.
J Virol, 2004. 78 ( 12 ): p . 6381-8 ), and there have been no E. & Zhang, F. A split- Cas9 architecture for inducible
established cases of pathology associated with AAV infec genome editing and transcription modulation . Nature bio
tion , making them one of the most promising vectors cur technology 33 , 139-142 (2015), Nihongaki, Y., Kawano , F.,
rently used in clinical trials. Moreover, tissue-targeting is Nakajima, T. & Sato , M.Photoactivatable CRISPR -Cas9 for
easily accomplished by pseudotyping to AAV serotypes with optogenetic genome editing . Nature biotechnology 33 , 755
suitable tropism . Of interest is AAV serotype 9 , which 760 (2015 ), Nishimasu , H. et al. Crystal Structure of Staphy
robustly transduces multiple cell types in the body (see lococcus aureus Cas9. Cell 162, 1113-1126 ( 2015 ), Truong,
Zincarelli, C., et al., Analysis of AAV serotypes 1-9 medi D. J. et al. Development of an intein -mediated split -Cas9
ated gene expression and tropism in mice after systemic system for gene therapy. Nucleic Acids Res 43 , 6450-6458,
injection .Mol Ther, 2008. 16 (6 ): p . 1073-80 . and Foust , K. doi: 10.1093 /nar /gkv601 ( 2015 ), and Fine, E. J. et al. Trans
D., et al., Intravascular AAV9 preferentially targets neonatal spliced Cas9 allows cleavage of HBB and CCR5 genes in
neurons and adult astrocytes. Nat Biotechnol, 2009.27 (1 ): p . human cells using compact expression cassettes. Scientific
59-65.) and crosses endothelial barriers (e.g. blood -brain reports 5 , 10777 (2015 ) .
barrier, see Foust, K. D., et al., Intravascular AAVO prefer [0073] According to one aspect, two portions of a Cas9
entially targets neonatal neurons and adult astrocytes. Nat protein are provided within a cell or are otherwise expressed
Biotechnol, 2009. 27 ( 1): p . 59-65 and Zhang, H. et al. within a cell and are combined together to form the Cas9
Several rAAV vectors efficiently cross the blood -brain bar protein . The two portions of the Cas9 protein are sufficient
rier and transduce neurons and astrocytes in the neonatal in length such that when they are combined into the Cas9
mouse central nervous system . Mol Ther, 2011 19 (8 ): p . protein , the Cas9 protein has the function of co -localizing at
1440-1448 ) that block access by other delivery vectors . a target nucleic acid with a guide RNA as described above .
Together, AAV and CRISPR present an enticing combina According to certain aspects , various methods known to
tion for achieving systemic gene -editing , but a key obstacle those of skill in the art may be used to combine the two or
has been the limited capacity of AAV for packaging exog more portions of a Cas9 protein together. Exemplary meth
enous sequences (< 4.7 kb). Dong, J. Y., P. D. Fan , and R. A. ods and linkers include split - intein protein trans-splicing for
Frizzell , Quantitative analysis of the packaging capacity of reconstituting the Cas9 protein as is known in the art and as
recombinant adeno - associated virus. Hum Gene Ther, 1996 . described herein . Other methods include protein -protein
7 (17 ): p . 2101-12 . Of the various Case orthologs that have interacting domains (see Zakeri, B., et al., Peptide tag
been co -opted for genome engineering (ST1 , Nm , Sa) (see forming a rapid covalent bond to a protein , through engi
Ran, F. A. et al. In vivo genome editing using Staphylococ neering a bacterial adhesion .Proc Natl Acad Sci USA , 2012 .
cus aureus Cas9 . Nature , 2015. 520 : p . 186-91 and Esvelt , 109 (12 ): p . E690-7 and Fierer , J. O., G. Veggiani, and M.
K. M., et al., Orthogonal Cas9 proteins for RNA - guided Howarth , SpyLigase peptide- peptide ligation polymerizes
gene regulation and editing. Nat Methods, 2013. 10 ( 11 ): p . affibodies to enhance magnetic cancer cell capture . Proc Natl
US 2019/0345483 A1 Nov. 14 , 2019
14
Acad Sci USA , 2014. 111 ( 13): p . E1176-81) or small group if present to the target nucleic acid so that the
molecule dependent interactions. See Los, G. V., et al., functional group can perform the desired function . A guide
HaloTag : a novel protein labeling technology for cell imag RNA having a spacer sequence length where the enzymati
ing and protein analysis . ACS Chem Biol, 2008. 3(6 ): p . cally active Cas9 will cut or nick the target nucleic acid may
373-82 and Keppler , A., et al., A general method for the be termed an “ enzymatic guide RNA ” to the extent that such
covalent labeling of fusion proteins with smallmolecules in a guide RNA facilitates enzymatic activity of the Cas9 . An
vivo . Nat Biotechnol, 2003. 21 (1): p . 86-9 ; Mootz, H. D., enzymatic guide RNA has an exemplary spacer sequence
Blum , E. S., Tyszkiewicz , A. B. & Muir , T. W. Conditional length of25 to 15 nucleotides. A guide RNA having a spacer
protein splicing: a new tool to control protein structure and sequence length where the enzymatically active Cas9 will
function in vitro and in vivo . J Am Chem Soc 125 , 10561 function as a nuclease null Cas9 and may be termed a
10569, doi: 10.1021/ ja0362813 (2003). “ nonenzymatic guide RNA ” to the extent that such a guide
[0074 ] Embodiments of the present disclosure are directed RNA will inhibit enzymatic activity of the Cas9. A nonen
to the use of a CRISPR /Cas system and, in particular, an zymatic guide RNA has an exemplary spacer sequence
enzymatically active Cas9 protein optionally having a func length of 16 to 8 nucleotides. It is to be understood that the
tional group attached thereto , and one or more guide RNAs enzymatically active Cas9 may still be referred to as such
which includes a spacer sequence , a tracr mate sequence and even though it is used with a nonenzymatic guide RNA and
a tracr sequence . According to certain aspects, a guide RNA where the enzymatically active Cas9 does not cut or nick the
which facilities enzymatic activity of the Cas9 protein has an target nucleic acid . The enzymatically active Cas9 can be
exemplary spacer sequence including between 25 and 15 programmed to cut or operate as a nuclease null Cas9 based
nucleotides in length . According to certain aspects, a guide on the selected spacer sequence length . It is to be understood
RNA which inhibits enzymatic activity of the Cas9 protein that for particular target nucleic acids, an exemplary enzy
has an exemplary spacer sequence including between 14 and matic guide RNA length or an exemplary nonenzymatic
8 nucleotides in length. According to certain methods, two guide RNA length may include 1 or two nucleotides outside
or more or a plurality of guide RNAs may be used in the of the exemplary ranges described herein .
practice of certain embodiments based on whether one of [0078 ] According to certain aspects , the tracr mate
skill desires the species of enzymatically active Cas9 protein sequence is between about 17 and about 27 nucleotides in
optionally having a functional group attached thereto to cut length. According to certain aspects , the tracr sequence is
or nick a desired nucleic acid or to deliver the functional between about 65 and about 75 nucleotides in length .
group to a desired nucleic acid so that the functional group According to certain aspects, the linker nucleic acid
can perform the function . sequence is between about 4 and about 6 .
[0075 ] The term spacer sequence is understood by those of [0079 ] The functional group or function conferring protein
skill in the art and may include any polynucleotide having or domain may be joined , fused , connected , linked or
sufficient complementarity with a target nucleic acid otherwise tethered such as by covalent hoods to the enzy
sequence to hybridize with the target nucleic acid sequence matically active Cas9 protein using methods known to those
and direct sequence -specific binding of a CRISPR complex of skill in the art.
to the target sequence . A CRISPR complex may include the [0080 ] Functional groups or function conferring proteins
guide RNA and the Cas9 protein . The guide RNA may be or domains within the scope of the present disclosure
formed from a spacer sequence covalently connected to a include transcriptional modulators or effector domains
tracr mate sequence (which may be referred to as a crRNA ) known to those of skill in the art. Suitable transcriptional
and a separate tracr sequence , wherein the tracr mate modulators include transcriptional activators . According to
sequence is hybridized to a portion of the tracr sequence . one aspect, the transcriptional regulator protein or domain
According to certain aspects , the tracrmate sequence and the upregulates expression of the target nucleic acid . Suitable
tracr sequence are connected or linked such as by covalent transcriptional modulators include transcriptional repres
bonds by a linker sequence , which construct may be referred sors. According to one aspect, the transcriptional regulator
to as a fusion of the tracr mate sequence and the tracr protein or domain downregulates expression of the target
sequence. The linker sequence referred to herein is a nucleic acid . Exemplary transcriptional activators include
sequence of nucleotides, referred to herein as a nucleic acid VP64 , VP16 , VP160, VP48 , VP96 , p65 , Rta , VPR , hsf1, and
sequence, which connect the tracr mate sequence and the p300 Suitable transcriptional repressors include KRAB .
tracr sequence . Accordingly, a guide RNA may be a two Transcriptional activators and transcriptional repressors can
component species ( i.e., separate crRNA and tracr RNA be readily identified by one of skill in the art based on the
which hybridize together ) or a unimolecular species ( i.e., a present disclosure
crRNA -tracr RNA fusion , often termed an sgRNA ). [0081 ] Functional groups, function conferring proteins or
[0076 ] Tracr mate sequences and tracr sequences are domains within the scope of the present disclosure include
known to those of skill in the art, such as those described in detectable groups or markers or labels . Such detectable
US 2014/0356958 . The tracr mate sequence and tracr groups ormarkers or labels can be detected or imaged using
sequence used in the present disclosure is N20 or more to methods known to those of still in the art to identify the
N8- gtatagagctagaaatagcaagttaaaataaggctagtccgttatcaactt location of the target nucleic acid sequence . Indirect attach
gaaaaagtggcaccgagtcggtgc with N20-8 being the number of ment of a detectable label or maker is contemplated by
nucleotides complementary to a target locus of interest . aspects of the present disclosure . Detectable labels or mark
[0077] According to certain aspects , the guide RNA spacer ers can be readily identified by one of skill in the art based
sequence length determines whether the enzymatically on the present disclosure. Detectable groups include fluo
active Cas9 optionally having a functional group attached rescent proteins such as GFP, RFP, BFP , EYFP, sfGFP,
thereto will function to cut or nick the target nucleic acid or mcherry, iRFP, citrine , morange, cerulean , mturquoise,
to act as a nuclease null Cas9 and deliver the functional EBFP, EBFP2 , Azurite, mKalamal, ECP, CYPET, mtur
US 2019/0345483 A1 Nov. 14 , 2019
15
quoise2, YFP, Venus, and Ypet and the like. Other useful nucleic acid in a cell comprising providing to the cell the
detectable groups include spytag , spycatcher , snap tags, enzymatically active Cas9 protein having the functional
biotin , streptavidin , and suntag and the like. group or moiety attached thereto and a guide RNA having
[0082 ] Functional groups, function conferring proteins or spacer sequence between 16 and 8 nucleotides in length
domains within the scope of the present disclosure include wherein the guide RNA and the Cas9 protein form a co
binding functional groups which may function to bind to localization complex with the target nucleic acid and where
desired molecules. Such binding functional groups include the enzymatically active Cas9 protein is rendered non
aptamers ms2 to MCP, pp7 to PCP, com to Com binding endonucleolytically active and where the functional group or
protein , inteins, FKBP to FRB , PMAG to nMAG and Cry2 moiety is delivered to the target nucleic acid . Methods
and the like . described herein can be performed in vitro , in vivo or ex
[0083 ] According to one aspect, embodiments described vivo . According to one aspect, the cell is a eukaryotic cell or
herein include guide RNA having a length including the sum a prokaryotic cell. According to one aspect, the cell is a
of the lengths of a spacer sequence , tracr mate sequence , bacteria cell, a yeast cell, a mammalian cell , a plant cell or
tracr sequence, and linker sequence ( if present). Accord an animal cell . According to one aspect , the Cas9 protein is
ingly, such a guide RNA may be described by its total length an enzymatically active Cas9 protein , a Cas9 protein wild
which is a sum of its spacer sequence , tracr mate sequence , type protein , or an enzymatically active Cas9 nickase .
tracr sequence, and linker sequence . According to this Additional exemplary Cas9 proteins include Cas9 proteins
aspect, all of the ranges for the spacer sequence , tracr mate attached to , bound to or fused with functional proteins such
sequence, tracr sequence, and linker sequence (if present) as transcriptional regulators, such as transcriptional activa
are incorporated herein by reference and need not be tors or repressors VPR , a Fok- domain , such a Fok 1, an
repeated . One of skill will readily be able to sum each of the aptamer, a binding protein , PP7 , MS2 and the like .
portions of a guide RNA to obtain the total length of the [0087 ] Embodiments of the present disclosure are directed
guide RNA sequence. Aspects of the present disclosure are to a method of delivering an enzymatically active Cas9
directed to methods of making such guide RNAs as protein optionally having a functional group attached thereto
described herein by expressing constructs encoding such to cells within a subject comprising administering to the
guide RNA using promoters and terminators and optionally subject, such as systemically administering to the subject,
other genetic elements as described herein . such as by intravenous administration or injection , intrap
[0084 ] According to certain aspects, the guide RNA and eritoneal administration or injection , intramuscular admin
the enzymatically active Cas9 optionally having a functional istration or injection , intracranial administration or injection ,
group attached thereto which interacts with the guide RNA intraocular administration or injection , subcutaneous admin
are foreign to the cell into which they are introduced or istration or injection , a enzymatically active Cas9 protein
otherwise provided .According to this aspect, the guide RNA optionally having a functional group attached thereto or a
and the enzymatically active Cas9 optionally having a nucleic acid encoding the enzymatically active Cas9 protein
functional group attached thereto are nonnaturally occurring optionally having a functional group attached thereto .
in the cell in which they are introduced , or otherwise [0088 ] Embodiments of the present disclosure are directed
provided . To this extent, cells may be genetically engineered to a method of delivering a guide RNA to cells within a
or genetically modified to include the CRISPR /Cas systems subject comprising administering to the subject , such as
described herein . systemically administering to the subject, such as by intra
[0085 ] One such CRISPR / Cas system uses the S. pyogenes venous administration or injection , intraperitoneal adminis
Cas9 nuclease (Sp. Cas9 ), an extremely high -affinity ( see tration or injection , intramuscular administration or injec
Sternberg, S. H., Redding , S., Jinek , M., Greene, E. C. & tion , intracranial administration or injection , intraocular
Doudna , J. A. DNA interrogation by the CRISPR RNA administration or injection , subcutaneous administration or
guided endonuclease Cas9. Nature 507, 62-67 (2014 ) hereby injection , a guide RNA or a nucleic acid encoding the guide
incorporated by reference in its entirety ), programmable RNA .
DNA -binding protein isolated from a type II CRISPR [0089 ] Embodiments of the present disclosure are directed
associated system (see Garneau , J. E. et al. The CRISPR /Cas to a method of delivering an enzymatically active Cas9
bacterial immune system cleaves bacteriophage and plasmid protein optionally having a functional group attached thereto
DNA . Nature 468, 67-71 ( 2010 ) and Jinek , M. et al. A and a guide RNA to cells within a subject comprising
programmable dual-RNA -guided DNA endonuclease in administering to the subject , such as systemically adminis
adaptive bacterial immunity. Science 337 , 816-821 (2012 ) tering to the subject, such as by intravenous administration
each of which are hereby incorporated by reference in its or injection , intraperitoneal administration or injection ,
entirety ). The DNA locus targeted by Cas9 precedes a three intramuscular administration or injection , intracranial
nucleotide (nt) 5'-NGG -3' “ PAM ” sequence, and matches a administration or injection , intraocular administration or
15-22 -nt guide or spacer sequence within a Cas9-bound injection , subcutaneous administration or injection , an enzy
RNA cofactor, referred to herein and in the art as a guide matically active Cas9 optionally having a functional group
RNA . Altering this guide RNA is sufficient to target Cas9 to attached thereto or a nucleic acid encoding the enzymati
a target nucleic acid . In a multitude of CRISPR -based cally active Cas9 protein optionally having a functional
biotechnology applications, the guide is often presented in a group attached thereto and a guide RNA or a nucleic acid
so - called sgRNA (single guide RNA ), wherein the two encoding the guide RNA .
natural Cas9 RNA cofactors (crRNA and tracrRNA ) are [0090 ] Methods of non - viral delivery of nucleic acids or
fused via an engineered loop . native DNA binding protein , native guide RNA or other
[0086 ] Embodiments of the present disclosure are directed native species include lipofection , microinjection , biolistics,
to a method of delivering a functional group or moiety virosomes, liposomes, immunoliposomes, polycation or lip
attached to a enzymatically active Cas9 protein to a target id :nucleic acid conjugates, naked DNA , artificial virions,
US 2019/0345483 A1 Nov. 14 , 2019
16
and agent-enhanced uptake of DNA . Lipofection is spersed short palindromic repeats (CRISPR ) transcripts ,
described in e.g. , U.S. Pat . Nos. 5,049,386 , 4,946,787 ; and proteins, enzymes, mutant forms thereof, fusion proteins
4,897,355) and lipofection reagents are sold commercially thereof, etc.).
( e.g., TransfectamTM and LipofectinTM ). Cationic and neutral [0092 ] Aspects of the methods described herein may make
lipids that are suitable for efficient receptor-recognition use of terminator sequences . A terminator sequence includes
lipofection of polynucleotides include those of Felgner, WO a section of nucleic acid sequence that marks the end of a
91/17424 ; WO 91/16024 . Delivery can be to cells ( e.g. in gene or operon in genomic DNA during transcription . This
vitro or ex vivo administration ) or target tissues ( e.g. in vivo sequence mediates transcriptional termination by providing
administration ). The term native includes the protein , signals in the newly synthesized mRNA that trigger pro
enzyme or guide RNA species itself and not the nucleic acid cesses which release the mRNA from the transcriptional
encoding the species . complex . These processes include the direct interaction of
the mRNA secondary structure with the complex and /or the
[0091 ] Regulatory elements are contemplated for use with indirect activities of recruited termination factors . Release of
the methods and constructs described herein . The term the transcriptional complex frees RNA polymerase and
“ regulatory element” is intended to include promoters , related transcriptional machinery to begin transcription of
enhancers, internal ribosomal entry sites (IRES ), and other new mRNAs. Terminator sequences include those known in
expression control elements ( e.g. transcription termination the art and identified and described herein .
signals, such as polyadenylation signals and poly - U [0093] Aspects of the methods described herein may make
sequences ). Such regulatory elements are described , for use of epitope tags and reporter gene sequences. Non
example , in Goeddel, GENE EXPRESSION TECHNOL limiting examples of epitope tags include histidine (His)
OGY: METHODS IN ENZYMOLOGY 185 , Academic tags, V5 tags, FLAG tags , influenza hemagglutinin (HA )
Press, San Diego , Calif. ( 1990 ). Regulatory elements tags, Myc tags, VSV-G tags, and thioredoxin ( Trx ) tags.
include those that direct constitutive expression of a nucleo Examples of reporter genes include , but are not limited to ,
tide sequence in many types ofhost cell and those that direct glutathione- S -transferase (GST ), horseradish peroxidase
expression of the nucleotide sequence only in certain host (HRP), chloramphenicol acetyltransferase (CAT) beta - ga
cells ( e.g., tissue -specific regulatory sequences ). A tissue lactosidase, betaglucuronidase, luciferase, green fluorescent
specific promoter may direct expression primarily in a protein (GFP ), HcRed , DsRed , cyan fluorescent protein
desired tissue of interest, such asmuscle, neuron, bone, skin ,
(CFP ), yellow fluorescent protein (YFP ), and autofluores
cent proteins including blue fluorescent protein (BFP ).
blood , specific organs ( e.g. liver ,pancreas), or particular cell (0094 ] The following examples are set forth as being
types (e.g. lymphocytes ). Regulatory elements may also representative of the present disclosure. These examples are
direct expression in a temporal-dependent manner, such as not to be construed as limiting the scope of the present
in a cell -cycle dependent or developmental stage -dependent disclosure as these and other equivalent embodiments will
manner, which may or may not also be tissue or cell-type be apparent in view of the present disclosure , figures and
specific . Regulatory elements may also direct expression in accompanying claims.
an inducible manner, such as in a small -molecule dependent
or light-dependent manner . In some embodiments , a vector Examples
may comprise one or more pol III promoter ( e.g. 1 , 2 , 3 , 4 , [0095 ] CRISPR - Cas9 holds tremendous promise in cor
5 , or more pol III promoters), one or more pol II promoters recting genetic defects , and its delivery by adeno -associated
( e.g. 1, 2 , 3 , 4 , 5 , ormore pol II promoters), one ormore pol viruses (AAVs ) is thought to be exceptionally safe . How
I promoters (e.g. 1, 2 , 3 , 4 , 5 , or more pol I promoters), or ever, immunological reactions against encoded transgenes
combinations thereof. Examples of pol III promoters and /or the viral capsid have sometimes been observed
include, but are not limited to , U6 and H1 promoters. (reviewed in Mays, L. E. & Wilson , J. M. The complex and
Examples of pol II promoters include, but are not limited to , evolving story of T cell activation to AAV vector- encoded
the retroviral Rous sarcoma virus (RSV ) LTR promoter transgene products. Mol Ther 19, 16-27, doi:10.1038 /mt.
(optionally with the RSV enhancer ), the cytomegalovirus 2010.250 (2011)). Hence , in this study, it has been sought to
(CMV ) promoter (optionally with the CMV enhancer ) (see , first establish a flexible AAV -CRISPR - Cas9 platform that
e.g., Boshart et al , Cell , 41: 521-530 (1985 )), the SV40 enables the wide spectrum of unrealized applications in
promoter , the dihydrofolate reductase promoter, the ß -actin vivo . Second , the host response to the system has been
promoter , the phosphoglycerolkinase (PGK ) promoter, and tracked . This is important because the exogenous nature of
the EFla promoter and Pol II promoters described herein . AAV -CRISPR -Cas9 might incite detrimental host reactions
Also encompassed by the term “ regulatory element” are against the encoded transgenes and/or viral capsid (reviewed
enhancer elements, such as WPRE; CMV enhancers; the in Mays, L. E. & Wilson , J. M. The complex and evolving
R -U5' segment in LTR of HTLV -I (Mol. Cell . Biol., Vol. story of T cell activation to AAV vector-encoded transgene
8 ( 1 ), p . 466-472 , 1988); SV40 enhancer; and the intron products. Mol Ther 19 , 16-27, doi:10.1038/mt.2010.250
sequence between exons 2 and 3 of rabbit B -globin (Proc . (2011 )). Understanding the host responses towards AAV
Natl. Acad . Sci. USA ., Vol. 78 (3 ), p . 1527-31, 1981 ). It will CRISPR -Cas9 would identify confounding factors that
impact experimental rigor, highlight relevant considerations
be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the for clinical translation , and provide a roadmap for engineer
choice of the host cell to be transformed , the level of ing efficient genome manipulation systems. Specifically, this
expression desired , etc. A vector can be introduced into host example describes the immunogenicity of AAV - CRISPR
cells to thereby produce transcripts , proteins, or peptides , Cas9 in mice, specifically that of AAV-split- Cas9 , a platform
including fusion proteins or peptides, encoded by nucleic capable of postnatal genome-editing , transcriptional regula
acids as described herein (e.g., clustered regularly inter tion , and further domain fusions. AAV elicits a humoral
US 2019/0345483 A1 Nov. 14 , 2019
17
immune response, inducing antibodies targeting motifs asso delivery of CRISPR system components in vivo . Nature
ciated with viral functions . Cas9 elicits both humoral and biotechnology , doi :10.1038 /nbt.3471 (2016 ); Senis , E. et al.
cellular immune responses, but its delivery by AAV miti CRISPR / Cas9 -mediated genome engineering: an adeno -as
gates overt tissue or cellular damage seen with alternative sociated viral (AAV ) vector toolbox. Biotechnology journal
delivery methods . This study provides the first demonstra 9 , 1402-1412, doi: 10.1002/biot.201400046 ( 2014 ); Swiech ,
tion of postnatal CRISPR -Cas9 applications beyond L. et al. In vivo interrogation of gene function in the
genome-editing, and elucidates the AAV -CRISPR - Cas9 mammalian brain using CRISPR - Cas9. Nature biotechnol
safety profile necessary for bringing it into the clinic . ogy 33 , 102-106 . doi: 10.1038 /nbt.3055 ( 2015 )) (pay load
[ 0096 ] Of the various CRISPR - Cas9 (Mali, P. et al. RNA limit s4.7 kb ). This obstacle is exacerbated with the most
guided human genome engineering via Cas9. Science 339, widely used , but larger SpCas9 (4.2 kb ), which makes
823-826 , doi: 10.1126 /science.1232033 (2013 ); Cong , L. et packaging of even the minimum functional cassette chal
al . Multiplex genome engineering using CRISPR /Cas sys lenging (Long . C. et. al. Postnatal genome editing partially
tems. Science 339, 819-823, doi: 10.1126 /science.1231143 restores dystrophin expression in a mouse model ofmuscu
(2013); Jinek , M. et al. A programmable dual-RNA -guided lar dystrophy. Science 351, 400-403, doi: 10.1126 / science .
DNA endonuclease in adaptive bacterial immunity . Science aad5725 (2016 ); Senis, E. et al . CRISPR / Cas9 -mediated
337, 816-821, doi: 10.1126 /science.1225829 (2012 ); Ran , F. genome engineering : an adeno -associated viral (AAV ) vec
A. et al. In vivo genome editing using Staphylococcus tor toolbox . Biotechnology journal 9, 1402-1412 , doi:10 .
aureus Cas9 . Nature 520 , 186-191 , doi: 10.1038 /na
1002/biot.201400046 (2014 ); Swiech , L. et al. In vivo
ture14299 (2015) ; Esvelt, K. M. et al. Orthogonal Cas9 interrogation of gene function in themammalian brain using
proteins for RNA -guided gene regulation and editing . CRISPR -Cas9. Nature biotechnology 33 , 102-106 , doi: 10 .
Nature methods 10 , 1116-1121 , doi: 10.1038/nmeth.2681 1038/nbt.3055 (2015 )). Resolving this challenge would
( 2013 )) and recently characterized CRISPR -Cpfi (Zetsche, enable novel applications currently unachievable.
B. et al. Cpfl is a single RNA -guided endonuclease of a [0097] Hence , in this example, it is sought to firstly ,
class 2 CRISPR -Cas system . Cell 163, 759-771, doi: 10 . combine AAV , SpCas9 , and fusion domains for a platform
1016 /j.cell.2015.09.038 (2015 )) orthologs, this example capable of postnatal genome-editing and epigenetic modu
chose to examine the Streptococcus pyogenes Cas9 (Sp lation , and secondly , to investigate the immunogenicity of
Cas9 ) due to multiple attractive features. SpCas9 has the the therapeutic modality .
least restrictive protospacer adjacent motif (PAM ) require [0098 ] To date, determined structures of Cas9 and Cpfi
ment, which fundamentally dictates the density of possible proteins generally adopt a bi-lobed architecture (SpCas9
target sites per given genome (FIG . 1A ). Conversely , more (Nishimasu , H. et al. Crystal structure of Cas9 in complex
restrictive PAM requirements (e.g., St1 (Esvelt, K. M. et al. with guide RNA and target DNA. Cell 156 , 935-949, doi:
Orthogonal Cas9 proteins for RNA-guided gene regulation 10.1016 / j.cell.2014.02.001 (2014 ); Jinek , M. et al. Struc
and editing. Nature methods 10 , 1116-1121, doi: 10.1038 / tures of Cas9 endonucleases reveal RNA -mediated confor
nmeth.2681 (2013 )); Nm (Esvelt, K. M. et al. Orthogonal mational activation . Science 343 , 1247997 , doi: 10.1126 /
Cas9 proteins for RNA -guided gene regulation and editing . science.1247997 (2014 )), SaCas9 (Nishimasu , H. et al.
Nature methods 10 , 1116-1121, doi: 10.1038 /nmeth.2681 Crystal Structure of Staphylococcus aureus Cas9. Cell 162,
( 2013 )); and Sa (Ran , F. A. et al. In vivo genome editing 1113-1126 , doi: 10.1016 / j.cell.2015.08.007 ( 2015 )), Ana
using Staphylococcus aureus Cas9. Nature 520 , 186-191, Cas9 (Jinek , M. et al. Structures of Cas9 endonucleases
doi: 10.1038/nature14299 ( 2015)) Cas9s ; As and Lb Cpfls reveal RNA -mediated conformational activation . Science
(Zetsche , B. et al. Cpfi is a single RNA - guided endonu 343, 1247997, doi: 10.1126 /science.1247997 (2014 )),
clease of a class 2 CRISPR -Cas system . Cell 163 , 759-771 , LbCpf1 ( Dong D , Ren K , Qiu X , Zheng J,Guo M , Guan X ,
doi:10.1016 / j.cell.2015.09.038 (2015 )) render significant Liu H , Li N , Zhang B , Yang D , Ma C , Wang S , Wu D , Ma
numbers of target sites inaccessible Combining SpCas9 with Y , Fan S , Wang J, Gao N , Huang Z , The crystal structure of
AAV is hence highly enticing to access the broadest target Cpfl in complex with CRISPR RNA , Nature , 2016 April 28;
ing range in vivo . Furthermore , the array of CRISPR - Cas9 532 ( 7600 ):522-6 ) AsCpfi ( Yamano T, Nishimasu H ,
applications beyond genome-editing has yet to be realized in Zetsche B , Hirano H , Slaymaker I M , Li Y , Fedorova I,
vivo , in part due to the large sizes of Cas9 transgenes that Nakane T , Makarova K S , Koonin E V , Ishitani R , Zhang F ,
leave little space for additional function -conferring elements Nureki O , Crystal Structure of Cpfl in Complex with Guide
within current AAV designs (Ran . F A et al. In vivo genome RNA and Target DNA , Cell, 2016 May 5 ; 165( 4 ): 949-62 ),
editing using Staphylococcus aureus Cas9. Nature 520 , but not FnCas9 (Hirano , H. et al. Structure and Engineering
186-191 , doi: 10.1038/nature14299 (2015 ); Nelson , C. E. et of Francisella novicida Cas9 . Cell 164. 950-961. doi: 10 .
al. In vivo genome editing improves muscle function in a 1016 / j.cell.2016.01.039 (2016 ))), offering the clue that they
mouse model of Duchenne muscular dystrophy. Science can be split into two well - folded halves. Indeed , previous
351, 403-407, doi: 10.1126 / science.aad5143 (2016 ); Tabe reports have demonstrated the feasibility of split - Cas9 ,
bordbar, M.et al. In vivo gene editing in dystrophic mouse albeit all in cell cultures and with different design principles
muscle and muscle stem cells . Science 351, 407-411 , doi: (Nishimasu , H. et al. Crystal Structure of Staphylococcus
10.1126 / science.aad5177 (2016 ); Long , C. et al. Postnatal aureus Cas9. Cell 162 , 1113-1126 , doi: 10.1016 / j.cell.2015 .
genome editing partially restores dystrophin expression in a 08.007 (2015 ); Zetsche, B., Volz , S. E. & Zhang, F. A
mouse model ofmuscular dystrophy. Science 351 , 400-403 , split - Cas9 architecture for inducible genome editing and
doi:10.1126 /science.aad5725 (2016 ); Yang, Y. et al. A dual transcription modulation . Nature biotechnology 33, 139
AAV system enables the Cas9 -mediated correction of a 142 , doi: 10.1038/nbt.3149 (2015 ); Wright, A. V. et al.Ratio
metabolic liver disease in newborn mice . Nature biotech nal design of a split - Cas9 enzyme complex . Proceedings of
nology , doi: 10.1038 /nbt.3469 (2016 ); Yin , H. et al. Thera the National Academy of Sciences of the United States of
peutic genome editing by combined viral and non -viral America 112 , 2984-2989 , doi: 10.1073 /pnas.1501698112
US 2019/0345483 A1 Nov. 14 , 2019
18
(2015 ); Nihongaki, Y., Kawano , F., Nakajima, T. & Sato , M. tripartite VPR ( Chavez, A. et al. Highly efficient Cas9
Photoactivatable CRISPR - Cas9 for optogenetic genome mediated transcriptional programming. Nature methods 12 ,
editing . Nature biotechnology 33 , 755-760 , doi: 10.1038/nbt. 326-328, doi: 10.1038/nmeth.3312 (2015 ))) for gene expres
3245 (2015 ); Truong , D. J. et al. Development of an intein sion upregulation (AAV -Cas9 -VPR ). This experiment fur
mediated split- Cas9 system for gene therapy. Nucleic Acids ther harnessed the findings that nuclease - active Cas9 can be
Res 43 , 6450-6458 , doi:10.1093/nar/gkv601 (2015 ); Fine, E. programmed with truncated gRNAs to bind genomic loci
J. et al. Trans-spliced Cas9 allows cleavage of HBB and without inducing DNA breaks, thereby allowing a single
CCR5 genes in human cells using compact expression Cas9 fusion protein to simultaneously effect genome-editing
cassettes. Scientific reports 5 , 10777 , doi: 10.1038 / and epigenetic regulation (Kiani, S. et al. Cas9 gRNA
srep10777 ( 2015 )). This example describes as an exemplary engineering for genome editing, activation and repression .
embodiment where SpCas9 is specifically split at its disor Nature methods 12 , 1051-1054 , doi: 10.1038 /nmeth.3580
dered linker between amino -acid residues V713 and D718 , (2015 ); Dahlman , J. E. et al. Orthogonal gene knockout and
hypothesizing that this maintains protein folding for each activation with a catalytically active Cas9 nuclease . Nature
lobe, such that the full -length Cas9 ( Cas9F1 ) can be recon biotechnology 33 , 1159-1161, doi: 10.1038/nbt.3390 (2015))
stituted seamlessly in vivo by split- intein protein trans (FIG . 1F ). Following transduction , nuclease - active AAV
splicing (Li, J., Sun, W., Wang. B., Xiao , X. & Liu , X. Q. Cas9 -VPR programmed with truncated gRNAs (14-15 nt
Protein trans-splicing as a means for viral vector-mediated spacers) upregulated gene expression ofthe targeted PD -L1,
in vivo gene therapy. Hum Gene Ther 19 , 958-964, doi: 10 . FST, and CD47 genes (FIG . 1G ) As with nuclease -inactive
1089/hum.2008.009 (2008 )) (FIG . 1B ). Thus, the Cas9 “dead ' Cas9FL (dCas9 )-activators (Chavez . A. et al . Highly
N -terminal lobe is fused with the Rhodothermus marinus efficient Cas9 -mediated transcriptional programming Nature
N -split -intein (Cas9N ) (2.5 kb ), and the C -terminal lobe is methods 12.326-328 . doi 10.1038 /nmeth.3312 ( 2015 ); Kon
fused with C -split - intein (Cas9c ) (2.2 kb) (FIGS . 2A and ermann , S. et al. Genome-scale transcriptional activation by
2B ), and each is designed to be individually packaged into an engineered CRISPR -Cas9 complex. Nature 517. 583
a separate AAV vector (FIG . 3A ), thereby liberating > 2 kb 588 , doi:10.1038 /nature14136 (2015)), gene activation by
within each AAV vector for additional elements . In trans AAV - Cas9 - VPR inversely correlated with the basal expres
fected cells , split - Cas9 was fully active , targeting all endog sion levels of the target genes (FIG . 1H ). It is noted that
enous genes tested at efficiencies 85 % to 115 % of Cas9FL when programmed with full -length gRNAs , AAV-Cas9
(FIG . 1C and FIGS. 2C and 2D ) . Full activity from struc VPR does not retain full endonucleolytic function of AAV
ture-guided split- intein reconstitution (Truong , D. J. et al . Cas9 (FIG . 1E ) Hence, split -Cas9 enables fusion of func
Development of an intein -mediated split -Cas9 system for tion - conferring domains within AAVg, and untethered split
gene therapy . Nucleic Acids Res 43, 6450-6458, doi: 10 . Cas9 offers more robust DNA - cleavage. Importantly , AAV
1093 /nar /gkv601 (2015 )) contrasts with sub -optimal activity Cas9FL design as previously described are unable to
from non -covalent heterodimerization (Nishimasu , H. et al. accommodate the 16 kb VPR domain fusion .
Crystal Structure of Staphylococcus aureus Cas9. Cell 162, [0100 ] To demonstrate functional in vivo , AAV -Cas9 .
1113-1126 , doi: 10.1016 / j.cell.2015.08.007 (2015 ); Zetsche , gRNAs targeting Msin was next pseudotyped to serotype 9
B., Volz , S. E. & Zhang, F. A split-Cas9 architecture for (AAV9- Cas9-gRNAsM3 -M4 ) and systematically delivered
inducible genome editing and transcription modulation . the viruses (5E11 or 4E12 vector genomes, vg ) into neonatal
Nature biotechnology 33 , 139-142 , doi:10.1038/nbt.3149 mice by intraperitoneal injection (FIG . 4A ).All AAV experi
(2015) ; Wright, A. V. et al. Rational design of a split- Cas9 ments in vivo were conducted in a randomized and double
enzyme complex . Proceedings of the National Academy of blind fashion . Deep -sequencing of whole tissues from
Sciences of the United States of America 112 , 2984-2989 , injected mice revealed a range of editing frequencies (up to
doi: 10.1073/pnas.1501698112 (2015); Nihongaki, Y., 10.9 % ), similar to those observed in cell culture (plasmid , up
Kawano, F., Nakajima, T. & Sato , M. Photoactivatable to 10.7 % . AAV, up to 6.3-24.6 % ). It was observed that
CRISPR - Cas9 for optogenetic genome editing. Nature bio editing frequencies exhibited inter-tissue bias for both on
technology 33 , 755-760, doi: 10.1038 /nbt.3245 (2015 )), sug target (Mstn ) and off- target (chr6-3906202 ) sites (FIGS. 5A
gesting that scarless protein ligation preserves Cas9 struc and 5B ), which suggests either absence of transduction m
ture and function . Next, Cas9C -P2A -turboGFP and Cas9N some cells, or that most cells were transduced but at different
U6 - gRNAs were packaged into AAV serotype DJ (AAV efficiencies. Infection assays argue against the first scenario ,
Cas9- gRNAs) (FIG . 3A ) and the viruses were to cultured because the AAV9 dosage used was in excess to transduce
cells . AAV -Cas9 - gRNAs modified all targeted genes in all examined cells within the liver,heart, and skeletalmuscle
differentiated myotubes (FIG . 1D , FIGS. 3B and 3C ), tail - tip (FIG . 5C ). Moreover, dual AAVIS co - transduced across
fibroblasts (FIG . 3D ) and spermatogonial cells (FIG . 1E ), most cells (FIG . 5D ), suggesting that gRNAs and split-Cas9
demonstrating robustness in three distinct cell types of co - deliver, and further supports the feasibility of applica
proliferative and terminally differentiated states . Therefore , tions involving multiple gRNAs (FIG . 5E) and Cas9 pro
split -Cas9 retains full activity of Cas9FL and opens the teins. The second scenario was then tested by measuring
spectrum of AAV-CRISPR - Cas9 applications previously viral concentration per cell , using qPCR to quantify AAV
unattainable , such as the programmable targeting of fusion vector genomes (vg ) per mouse diploid genome (dg). Con
domains towards DNA .
sistent with prior findings (Zincarelli, C., Soltys, S., Rengo ,
[0099 ] CRISPR - Cas9 -mediated epigenetic regulation has G. & Rabinowitz , J. E. Analysis of AAV serotypes 1-9
not been demonstrated in mice , but this ability would mediated gene expression and tropism in mice after systemic
address a whole spectrum of human epigenetic diseases injection . Mol Ther 16 , 1073-1080 , doi :10.1038 /mt.2008.76
irresolvable by genome-editing . Hence the next experiment ( 2008 )), AAVO showed preferential tropism for liver, heart
capitalized on the extra viral capacity of AAV-split-Cas9 to and skeletal muscle (vg/ dg of 850 , 370, 140 , respectively),
incorporate transcription -activator fusion domains ( 1.6 kb while lower vg /dg were detected in the brain and gonads
US 2019/0345483 A1 Nov. 14 , 2019
19
(FIG . 5F ). Strikingly, gene- editing frequencies correlated gen -specific T-cell clonotype common among injected mice
strongly with AAV vector copies (FIG . 4B , FIGS. 5G and ( i.e., public response), and with the remaining T-cell infil
5H ), indicating that delivery efficiency dictates mutation trates largely dissimilar between individuals ( i.e., private
rate . Therefore, despite sufficiency in infecting most cells , response ).
higher AAV9-Cas9 - gRNA copies per cell continue to [0104 ] To map Cas9 antibody epitopes, serum from each
increase editing rates. animal was co -incubated with M13 phage display libraries
[0101] Next, to demarcate its spatial biodistribution , tiling the Cas9 transgene, and antibody targets were deter
AAV9- Cas9- gRNAs activity was tracked at single -cell reso mined by Ig :phage pull-down (FIG . 8E , FIG . 9A ). Epitope
lution , using the Ai9 mouse line (Madisen , L. et al. A robust mapping shows that individual Cas9 -experienced animals
and high -throughput Cre reporting and characterization sys exhibit an antibody repertoire targeting unique residues of
tem for the whole mouse brain . Nat Neurosci 13 , 133-140 , Cas9 ; but three linear epitopes were observed more than
doi:10.1038 /nn.2467 (2010 )) that accurately couples once (FIG . 8F ). 1352 -ITGLYETRI-1360 consists ofresidues
genomic excision of a 3xStop cassette with tdTomato fluo recognizing gRNA stem loop 2 (Nishimasu , H. et al . Crystal
rescence activation . Systemic delivery of AAV9- Cas9 -gR structure of Cas9 in complex with guide RNA and target
NASTAL + TAR ( 5E11 or 4E12 vg) targeting the 3xStop cassette DNA. Cell 156 , 935-949, doi: 10.1016 / j.cell.2014.02.001
generated excision -dependent tdTomato + cells in all exam (2014 )); 122 - IVDEVAYHEKYP - 133 resides in the REC1
ined organs (FIG . 4C and FIG . 6 ). Targeted cells were domain also contributing to Cas9 :gRNA interactions, but do
widespread in the liver, heart and skeletal muscle (FIG . 7), not cover residues mediating the contact (Nishimasu , H. et
arguing against clonal descent and suggesting multiple inde al. Crystal structure of Cas9 in complex with guideRNA and
pendent targeting events . Gene- edited cell clusters were target DNA . Cell 156 , 935-949, doi: 10.1016 / j.cell.2014.02.
detected infrequently in the brain and gonad (< 0.001% of 001 (2014 )); 1126 -WDPKKYGGFD -1135 resides in the
cells) (FIG . 4C ), at a rate that evades detection with deep PAM -binding loop and contains conserved residues, but
sequencing of bulk samples (sensitivity limits ~ 0.2 % ). maintains wild -type Cas9 endonucleolytic function when
Hence , AAV9- Cas9 - gRNAs is robust in mice , and its wide selectively mutated ( 1125 -DWD-> AAA (Jinek , M. et al.
spread biodistribution also highlights that evaluation of Structures of Cas9 endonucleases reveal RNA -mediated
CRISPR - Cas9 tissue -level off-targeting should complement conformational activation . Science 343 , 1247997 , doi: 10 .
that at the genomic -level, which has so far been the primary 1126 /science.1247997 ( 2014 )), or D1135E (Kleinstiver, B.
focus. P. et al. Engineered CRISPR -Cas9 nucleases with altered
[0102] Furthermore , the platform enables transcriptional PAM specificities. Nature 523, 481-485 , doi: 10.1038/na
regulation in vivo .Mice were intramuscularly injected with ture14592 (2015 )) for increasing Cas9 specificity ). Combin
the same dosage of AAV9 - Cas9 - VPR - gRNAs, varying only ing these residue changes retain Cas9 activity (FIG . 9B ).
the spacer sequences to target different sets of genes. The This result demonstrates that identified antigenic protein
targeted PD - L1 and CD47 genes were activated by 2 - fold regions can be altered away from the original immunogenic
and 1.6 - fold respectively , as determined by qRT-PCR and amino acid sequences . Future large - scale functional variant
total mRNA sequencing (FIGS. 4D and 4E ). This demon profiling could reveal more residues amenable to epitope
strates , for the first time, postnatal transcriptional regulation recoding , so as to derive less immunogenic Cas9 and Cpf1
with CRISPR - Cas9 . proteins.
[0103 ] Having validated the AAV-split- Cas9 platform , it [0105 ] Unlike that with Cas9 , AAV9 elicited capsid -spe
was next to examine its immunogenicity , comparing it in cific antibodies (FIG . 9C ) against epitopes that were shared
parallel with intramuscular DNA electroporation . Following among injected animals at surprisingly high degrees (FIG .
expression of Cas9 in the adult tibialis anteriormuscle (FIG . 8G ), reminiscent of a public response to viruses recently
8A ), the draining lymph nodes enlarged with increased cell observed also in humans (Xu , G. J. et al. Viral immunology.
counts (FIG . 8B ). Cellular infiltration was largely absent in Comprehensive serological profiling of human populations
the lymph nodes of mice administered the same vectors using a synthetic human virome. Science 348 , aaa0698 , doi:
without the Cas9 coding sequence, indicating a Cas9 -driven 10.1126 / science.aaa0698 (2015 )). Epitope -mapping pro
immune response . T -cells orchestrate antigen - specific vides intriguing support that AAVO antigenicity derives from
immune responses , with each clonal lineage expressing a biophysical and functional aspects, instead of purely
unique T-cell receptor B -chain ( TCR - B ) CDR3 motif that sequence - level motifs . The metastable VP1 unique and
mediates most of the antigen - contact. To identify T-cell(s) VP1/2 common regions are antigenic, suggesting their exter
mediating the cellular response , the TCR -B repertoires of nalization from the viral interior for antigen capture. Immu
lymphocytes infiltrating the draining lymph nodes were nodominant epitopes in VP3 lie predominantly on the capsid
sequenced . It was observed that Cas9 -exposure stimulated surface (FIG . 9D ). Notably , while many of these residues
skewed expansion of T-cell clonotypic subsets (FIG . 8C ) , can be separately double -alanine mutated withoutdisrupting
which implies antigen -specific T-cell activation and prolif viral assembly (Adachi, K., Enoki, T.,Kawano, Y., Veraz,M.
eration . Four TCR - ß clonotypes were common to all Cas9 & Nakai, H. Drawing a high - resolution functional map of
experienced animals (n = 4 ) and undetected in all unexposed adeno -associated virus capsid by massively parallel
animals (n = 8). Because bona fide T-cells would proliferate sequencing. Nat Commun 5 , 3075 , doi : 10.1038 /
with antigen recall, itwas sought to confirm antigen -specific ncomms4075 (2014 )), they are predominantly implicated
T -cell expansion by challenging extracted lymphocytes with (Adachi, K., Enoki, T., Kawano , Y., Veraz, M. & Nakai, H.
purified Cas9 protein . Ofthe four initial clonotypes, one was Drawing a high -resolution functionalmap of adeno -associ
narrowed down on (VB16 , CDR3 : CASSLDRGQDTQYF ) ated virus capsid by massively parallel sequencing . Nat
as a true Cas9 -responsive T-cell clonotype, which prolifer Commun 5 , 3075 , doi: 10.1038 /ncomms4075 (2014 )) in
ated according to Cas9 protein restimulation (FIG . 8D ). maintaining viral blood persistency (FIG . 8H ) and liver
Hence, Cas9 elicits cellular immunogenicity, with an anti selective tropism (FIG . 8I ). Together , the immunodominant
US 2019/0345483 A1 Nov. 14 , 2019
20
VP3 epitopes (372 - FMIPQYGYLTLNDGSQAVG - 390 , muscle and muscle stem cells. Science 351 , 407-411, doi:
436 -MNPLIDQYLY -445 , and 494 - TQNNNSEFAWPG 10.1126 / science.aad5177 (2016 ); Long , C. et al. Postnatal
505 ) cover 12/18 of these residues associated with AAVO genome editing partially restores dystrophin expression in a
hepatotropism . Hence , AAVO elicits humoral immunogenic mouse model ofmuscular dystrophy . Science 351, 400-403 ,
ity that overlaps among all animals, across substantial doi:10.1126 / science.aad5725 (2016 ); Yang , Y. et al. A dual
regions of the capsid protein that modulate viral biodistri AAV system enables the Cas9 -mediated correction of a
bution . metabolic liver disease in newborn mice . Nature biotech
[0106 ] To obtain a better understanding of the immuno nology , doi: 10.1038 /nbt.3469 (2016 ) ; Yin , H. et al. Thera
logical reaction , mRNA sequencing was next conducted on peutic genome editing by combined viral and non -viral
tissues from AAV9-Cas9 -VPR - gRNAs and AAV9-turboRFP delivery of CRISPR system components in vivo . Nature
treated mice (4E12 and 1E11 vg), and the whole transcrip biotechnology , doi: 10.1038/nbt.3471 ( 2016 ); Senis, E. et al.
tomes were compared against those from mice treated with CRISPR / Cas9 -mediated genome engineering: an adeno -as
AAV9- turboRFP only (1E11 vg). Consistent with a change sociated viral (AAV ) vector toolbox . Biotechnology journal
in tissue composition following immune cell infiltration , 9, 1402-1412 , doi: 10.1002/biot.201400046 (2014 ); Swiech ,
differentially expressed genes are significantly enriched for L. et al. In vivo interrogation of gene function in the
immunological gene ontology processes. CD8 + T-cells are mammalian brain using CRISPR -Cas9 . Nature biotechnol
particular intriguing, because they effect tissue damage by ogy 33 , 102-106 , doi: 10.1038 /nbt.3055 (2015 )). This plat
cytolysis. However , a closer look at genes encoding key form enables in vivo applications including and beyond that
differentiation signals (e.g. IL - 12, Ifn -a /ß , IL -2 ) and of genome-editing, which has so far only begin to demon
cytolytic effector proteins (e.g. Prfl, Gmzb , FasL ) revealed strate the power of CRISPR -Cas9 in manipulating live
that these were not altered at statistically significant levels. mammals .
This led to examining functional readouts more sensitively [0109 ] The fundamental consideration for clinical imple
by intra - tissue immunofluorescence and histology . mentation of AAV- CRISPR - Cas9 lies in its safety . Alterna
[0107 ] Interleukin - 2 (IL - 2 ) is pivotal for cytolytic T-cell tive delivery methods such as DNA electroporation and
differentiation (Pipkin , M. E. et al. Interleukin - 2 and inflam adenoviruses (Wang , D. et al. Adenovirus-Mediated Somatic
mation induce distinct transcriptional programsthat promote Genome Editing of Pten by CRISPR /Cas9 in Mouse Liver in
the differentiation of effector cytolytic T cells. Immunity 32, Spite of Cas9 -Specific Immune Responses. Hum Gene Ther
79-90 , doi: 10.1016 / j.immuni.2009.11.012 (2010 )). Down 26 , 432-442, doi: 10.1089 /hum.2015.087 ( 2015 )) cause
stream , perforin (Prfl) is the essential pore- forming protein severe inflammation and immunological reactions within the
released by cytolytic T lymphocytes and NK cells to destroy host. Inherently benign delivery vectors are hence particu
target cells . In line with mRNA sequencing , immunofluo larly attractive . Precedents of immune evasion /tolerance by
rescence indicated minimal IL - 2 or perforin protein secre viruses have been documented in experimental and endog
tion within AAV9 - Cas9 - VPR - gRNAs-injected muscles enous settings, mediated by processes ranging through
(FIG . 10A ). Absence of elevated perforin release suggests insufficient activation, sub - threshold inflammatory milieu ,
minimal downstream cytolysis. Consistent with this, there and/or active immunosuppression (Mays , L. E. & Wilson , J.
was an absence of overt histological damage (FIG . 10C ), in M. The complex and evolving story of T cell activation to
stark contrast to that seen with DNA electroporation . IL -2 AAV vector-encoded transgene products. Mol Ther 19 ,
and perforin were both strongly elevated in muscles elec 16-27 , doi: 10.1038 /mt.2010.250 (2011); Zajac, A. J. et al.
troporated with Cas9 -encoding DNA and myofiber degen Viral immune evasion due to persistence of activated T cells
eration -repair clearly visible (FIGS. 10B and 10D ). This without effector function . J Exp Med 188 , 2205-2213
cellular damage is not due to simple physical disruptions (1998 ); Curtsinger, J. M., Lins, D. C. & Mescher, M. F.
from DNA electroporation , because IL2 and perforin levels Signal 3 determines tolerance versus full activation of naive
and myofiber cellular damage were significantly increased CD8 T cells: dissociating proliferation and development of
over that in vehicle -electroporated muscles and could be effector function . J Exp Med 197 , 1141-1151 , doi: 10.1084 /
alleviated by the administration of immunosuppressant jem.20021910 ( 2003 )). This example shows that AAV
( FK506 ), indicating an immunological basis. Hence, CRISPR -Cas9 activates immune responses without overt
although AAV - CRISPR - Cas9 activates the host immune cellular damage . This attests to its comparatively favorable
system , it does not trigger overt cellular damage observed safety profile , both for use in research models and ultimately
with alternative delivery methods. human patients.
[0108 ] CRISPR -Cas9 allows user -defined DNA -RNA [0110 ) Constructs and sequences. U6 -driven gRNA plas
protein interactions, driving a wide range of applications mids were constructed as described (Mali, P. et al. RNA
that includes epigenetic regulation and protein -complex guided human genome engineering via Cas9. Science 339,
recruitment. The use of CRISPR -Cas9 in vivo has the 823-826 , doi: 10.1126 /science.1232033 (2013 )). AAV plas
potential not just to correct genetic defects, but also to mid backbone was derived from pZac2.1 - CASI-EGFP
modulate the epigenome. Split - Cas9 shortens the coding RGB, a gift from Luk Vandenberghe .Minicircles parental
sequence below that of all known Cas9 orthologs, allowing plasmids were cloned in ZYCY10P3S2T, and minicircles
facile domain - fusions that AAV -Cas9FLs are unable to were generated as described (Kay, M. A., He, C. Y. & Chen ,
accommodate in their current forms (Ran , F. A. et al. In vivo Z. Y. A robust system for production of minicircle DNA
genome editing using Staphylococcus aureus Cas9. Nature vectors . Nature biotechnology 28 , 1287-1289 , doi: 10.1038 /
520 , 186-191, doi: 10.1038 /nature14299 (2015 ) ; Nelson , C. nbt.1708 ( 2010 )) . AAV plasmids were cloned in Stb13 (Life
E. et al. In vivo genome editing improvesmuscle function in Technologies C7373-03 ). All other plasmidswere cloned in
a mouse model of Duchenne muscular dystrophy. Science DH5a (NEB C2987H ). Protein transgenes were expressed
351, 403-407 , doi: 10.1126 /science.aad5143 (2016 ); Tabe from ubiquitous hybrid promoters: SMVP promoter (gener
bordbar, M. et al. In vivo gene editing in dystrophic mouse ated by fusing SV40 enhancer-CMV-promoter-chimeric
US 2019/0345483 A1 Nov. 14 , 2019
21
intron ),CASI promoter (Balazs, A. B. et al . Antibody -based [0114 ] For purification of AAV via chloroform -ammo
protection against HIV infection by vectored immunopro nium sulfate precipitation , 110th volume of chloroform and
phylaxis . Nature 481, 81-84 , doi: 10.1038 /nature10660 NaCl (1 M final concentration ) was added to the lysate and
(2012 )), or CAG promoter (Matsuda , T. & Cepko , C. L. shaken vigorously . After centrifugation , the supernatant was
Electroporation and RNA interference in the rodent retina in incubated with PEG - 8000 ( 10 % final w /v ) on ice for 21 hr
vivo and in vitro . Proceedings of the National Academy of or overnight. PEG -precipitated virions were centrifuged
Sciences of the United States of America 101, 16-22 , doi: (4000 g, 30 min ., 4° C.), and resuspended in 50 mM HEPES
10.1073 /pnas.2235688100 (2004 )). SMVP plasmid was buffer (PH8). 50 U /mlof Benzonase ( Sigma-Aldrich ) and 1
derived from PMAXGFP (Lonza ). PCAG -GFP was a gift U /ml of Riboshredder (Epicentre ) were added and incubated
from Connie Cepko (Addgene plasmid # 11150 ). PAAV for 30 min . at 37 ° C. An equal volume of chloroform was
CMV -HI- EGFP - Cre - WPRE -SV40pA was obtained from the then added , and vigorously vortexed . After centrifugation ,
University of Pennsylvania Vector Core. the aqueous phase was collected and residual chloroform
[0111] AAV Packaging and Purification . evaporated for 30 min . Ammonium sulfate precipitation of
[0112] AAVs were packaged via the triple -transfection AAVswere performed with a 0.5 M -2 M cut-off. AAVswere
method (Grieger, J. C., Choi, V. W. & Samulski, R. J. then resuspended and dialyzed in 1xPBS + 35 mM NaCl,
Production and characterization of adeno - associated viral quantified for viral titers , and stored in -80 ° C.
vectors . Nat Protoc 1, 1412-1428 , doi: 10.1038/nprot.2006 . [0115 ] All experiments with purified AAVs utilized the
207 (2006 ); Zolotukhin , S. et al. Recombinant adeno -asso iodixanol density gradient ultracentrifugation purification
ciated virus purification using novel methods improves method (Grieger , J. C., Choi, V. W. & Samulski, R. J.
infectious titer and yield . Gene Ther 6 , 973-985 , doi: 10 . Production and characterization of adeno -associated viral
1038 / sj.gt.3300938 ( 1999 )). HEK293 cells ( Cell Biolabs
AAV - 100 or Agilent 240073) were plated in growth media vectors . Nat Protoc 1, 1412-1428 , doi: 10.1038 /nprot.2006 .
207 ( 2006 ); Zolotukhin , S. et al. Recombinant adeno -asso
consisting of DMEM + glutaMAX + pyruvate + 10 % FBS (Life ciated virus purification using novel methods improves
Technologies), supplemented with 1xMEM non-essential
amino acids (Gibco ). Confluency at transfection was infectious1038 /
titer and yield . Gene Ther 6 , 973-985 , doi: 10 .
sj.gt.3300938 ( 1999 )), unless otherwise stated . The
between 70-90 % . Media was replaced with fresh pre collected AAV supernatant was first treated with 50 U /ml
warmed growth media before transfection . For each 15 - cm Benzonase and 1 U /ml Riboshredder for 30 min . at 37 ° C.
dish , 20 ug of pHelper (Cell Biolabs ), 10 ug of pRepCap After incubation , the lysate was concentrated to <3 ml by
[ encoding capsid proteins for AAV-DJ (Cell Biolabs ) or ultrafiltration with Amicon Ultra - 15 (50 kDa MWCO ) (Mil
AAV9 (UPenn Vector Core)], and 10 ug of PAAV were lipore ), and loaded on top of a discontinuous density gra
mixed in 500 ul of DMEM , and 200 ug of PEI “MAX ” dient
(Polysciences ) (40 kDa , 1 mg/ml in H ,O , pH 7.1) added for Optiprepconsisting
(Sigma
of 2 ml each of 15 % , 25 % , 40 % , 60 %
- Aldrich ) in an 11.2 ml Optiseal polypro
PEI:DNA mass ratio of 5 : 1 . The mixture was incubated for pylene tube (Beckman -Coulter ). The tubes were ultracentri
15 min ., and transferred drop -wise to the cell media . For
large- scale AAV production , HYPERFlask ‘ M ' (Corning ) fuged at 58000 rpm , at 18 ° C., for 1.5 hr, on an NVT65 rotor.
were used , and the transfection mixture consisted of 200 ug (pH 7.2%) supplemented
The 40 fraction was extracted , and dialyzed with 1xPBS
of pHelper, 100 ug of pRepCap , 100 ug of PAAV , and 2 mg Ultra -15 (50 kDa or 100withkDa35 MWCO mM NaCl, using Amicon
) (Millipore ). The
ofPEIMAX . The day after transfection , media was changed purified AAVs were quantified for viral titers , and stored in
to DMEM + glutamax +pyruvate + 2 % FBS. Cells were har -80 ° C.
vested 48-72 hrs after transfection by scrapping or disso [0116 ] AAV2/9 -CMV-HI-EGFP -Cre -WPRE -SV40 (Lot
ciation with 1xPBS (pH7.2 ) +5 mM EDTA , and pelleted at V4565MI -R ), AAV2 / 9 - CB7 -CI-EGFP -WPRE -rBG [Lot
1500 g for 12 min . Cell pellets were resuspended in 1-5 ml CS0516 ( 293 )], AAV2/9 -CB7 -CI-mCherry -WPRE -rBG (Lot
of lysis buffer (Tris HCl pH 7.5 + 2 mM MgCl+ 150 mM
NaCl), and freeze - thawed 3x between dry -ice -ethanol bath V4571MI- R ), and AAV2/ 9 -CMV -turboRFP -WPRE -rBG
(Lot V4528MI-R -DL) were obtained from the University of
and 37 ° C. water bath . Cell debris was clarified via 4000 g Pennsylvania
for 5 min ., and the supernatant collected . Downstream Vector Core .
processing differed depending on applications. [0117 ] AAV titers (vector genomes) were quantified via
[0113] For preparation of AAV -containing lysates, the hydrolysis -probe qPCR (Aurnhammer, C. et al. Universal
collected supernatant was treated with 50 U /ml of Benzo real-time PCR for the detection and quantification of adeno
nase (Sigma- Aldrich ) and 1 U /mlof Riboshredder (Epicen associated virus serotype 2 -derived inverted terminal repeat
tre ) for 30 min . at 37 ° C. to remove unpackaged nucleic sequences . Human gene therapy methods 23, 18-28 , doi: 10 .
acids, filtered through a 0.45 um PVDF filter (Millipore ), 1089/hgtb.2011.034 (2012)) against standard curves gener
and used directly on cells or stored in -80 ° C. ated from linearized parental AAV plasmids.
TABLE 1
A list of CRNA spacer sequences used in this study .
Spacer gRNAs set ( for
Sp CRNAS sequence , including 5 G from U6 promoter AAV9 - Cas9 - VPR )
Acvr2b B1 GGGCCATGTGGACATCCATGAGGTGAGACAGTGCCAGCGT
Acvr2b B3 GGCCTGAAGCCACTACAGCTGCTGGAGATCAAGGCTCG
Acvr2a A3 GGCCCTAGCATCTAAGTTCTCGCAGGC
US 2019/0345483 Al Nov. 14 , 2019
22
TABLE 1 - continued
A list of RNA spacer sequences used in this study .
Spacer gRNAs set ( for
Sp gRNAs sequence , including 5 ' G from U6 promoter AAV9 - Cas9 - VPR )
Acvr2a A4 GGTCATTCCATCTCAGCTGTGACAGCAGCGCAGAA
Mstn M3 GTCAAGCCCAAAGTCTCTCCGGGACCTCTT 1 and 2

Mstn M4 GGAATCCCGGTGCTGCCGCTACCCCCTCA 1 and 2
Ai9 Td5 GCTAGAGAATAGGAACTTCTT

Ai9 TAL GAAAGAATTGATTTGATACCG
Ai9 Td3 GATCCCCATCAAGCTGATC
Ai9 TAR GGTATGCTATACGAAGTTATT
PD - L1 P1 GCTCGAGATAAGACC 1
PD - L1 P2 GCTAAAGTCATCCGC 1
FST F1 GGTTCTTATTTGCGT
FST F2 GGAAATCAAAGCGGC 1 and 2
CD47 C1 GAAGGAGTTCCTCGG 1
CD47 C2 GAGGAGGTCCACTTC 1
TABLE 2
A list of locus - specific genotyping primers for deep - sequencing used in this study .
Target locus Sequence
Acvr2b F CTTTCCCTACACGACGCTCTTCCGATCTNNNNNNCTGGAGTGTTAGAGTGGGCG
Acvr2b R GGAGTTCAGACGTGTGCTCTTCCGATCTGACTGCCCCATGGAAAGACA
Mstn F CTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGGGCCATGAAAGGAAAAATGAAGT
Mstn R GGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCTGGGGTTTGCTTGGT
Acvr2a F CTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGAGATATAAGCTGAATAAGGCCAATGACATACT
Acvr2a R GGAGTTCAGACGTGTGCTCTTCCGATCTCTACTGCTCTTTCCTGCCGA
TABLE 3 TABLE 3 - continued

A list of qPCR probes and primers used in this A list of qPCR probes and primers used in this
study . study .
Target locus Sequence Target locus Sequence
Acyr2b F GCCTACTCGCTGCTGCCCATT AAV ITR F GGAACCCCTAGTGATGGAGTT
Acvr2b R CCTGGAGACCCCCAAAAGCTC
AAV ITR R CGGCCTCAGTGAGCGA
Acvr2b probe /5HEX / AGATCT + TC + CC + AC + TT + CA +GGT /
3IABkFQ / AAV ITR probe /56-FAM / CACTCCCTCTCTGCGCGCTCG / 3BH
Coding sequences of split - Cas9 .

Coding sequence for SpCas9N - RmaIntN :
MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR
ICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG
US 2019/0345483 A1 Nov. 14 , 2019
23
- continued
DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL
SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVROQLPEKYKEIFFDOSKNGYA
GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKORTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGP
LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI
VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL
FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVCLAGDTLITLADGRRVPIRELVS
QONFSVWALNPOTYRLEARVSRAFCTGIKEVYRLTTRLGRSIRATANHRFLTPOGWKRVDELOPGDYIALPRRIPTAS .
ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACTCCATTGGGCTCGATAT
CGGCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATA
CCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAAACGGCCGAAGCCACGCGGCT
CAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGG
CTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCA
ATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGAC
AGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAG
GGGGACCTGAACCCAGACAACACGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAG
AACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCT
CATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACT
TTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGC
TGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATT
CTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTT
GACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTA
CGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCAC
CGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACC
AGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGA
TTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTC
GCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAA
AGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTT
ATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCT
ATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTT
TCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTA
AAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATA
GGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGAT
ATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTT
CTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAG
AAAGCACAAGTTTGTCTGGCTGGCGATACTCTCATTACCCTGCCCGATGGACGACGAGTGCCTATTAGAGAACTGGTGT
CACAGCAGAATTTTTOCGTGTGGGCTCTGAATCCTCAGACTTACCGCCTGGAGAGGGCTAGAGTGAGTAGAGCTTTCTG
TACCGGCATCAAACCTGTGTACCGCCTCACCACTAGACTGGGGAGATCCATTAGGGCCACTGCCAACCACCGATTTCTC
ACACCTCAGGGCTCGAAACGAGTCGATGAACTCCAGCCTGGAGATTACCTGGCTCTGCCTAGGAGAATCCCTACTGCCT
CCTGA
Coding sequence for RmaIntC - SpCas9C - P2A -turboGFP :
MAAACPELROLAOSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVANDIJAHNSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK
PENIVIEMARENOTTOKGOKNSRERMKRIEEGIKELGSQILKEHPVENTOLONEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD
DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETRQITKHVAQILDSRMNTKY
DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF
FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG
APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVSRAGSGATNFSLLKQAGDVEENPGPMPAMKIECR
ITGTLNGVEFELVGGGEGTPEQGRMTNKNKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSY
RYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGYYSFVVDSHMHFKSAIHPSILONGGPMFAFRRV
EELHSNTELGIVEYQHAFKTPIAFARSRAR .
GGAACTGCGTCAGOGGAACTGCGTCAGCTGGCGCAGAGCGATGTGTATTGGGATCCGATTGTGAGCATTGAACCG
GATGGCGTGGAAGAAGTGTTTGATCTGACCGTGCCGGGCCCGCATAACTTTGTGGCGAACGATATTATTGCGCATAACT
CTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACC
GTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAA
CCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCC
AAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGG
ACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCA
AAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGT
TGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAA
GGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCA
AGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTA
TTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCA
TGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTAC
GGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTC
TTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAA
CAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCA
GGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACA
AGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGG
TTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGA
TCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTC
CCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAG
CTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATG
US 2019/0345483 A1 Nov. 14 , 2019
24
- continued
AGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAG
TGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCA
GAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGA
AAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATC
GACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGTCTCGAGCTGGATCCGGAGCCA
CGAACTTCTCTCTGTTAAAGCAAGCAGGGGACGTGGAAGAAAACCCCGGTCCTATGCCCGCCATGAAGATCGAGTGCC
GCATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCGAGCAGGGCCGCAT
GACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTT
CTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACAC
CCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCG
GCGACTTCAAGGTGGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCA
CCGTGGAGCACCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACGGC
GGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGC
CCCATGTTCGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTC
AAGACCCCCATCGCCTTCGCCAGATCTCGAGCTCGATGA
Coding sequence for Rma IntC - SpCas9C - 3PR :
MAAACPELROLAOSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVANDIIAHNSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK
PENIVIEMARENOTTOKGQKNSRERMKRIEEGIKELGSQILKEHPVENTOLONEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKD
DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITORKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY
DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYF
FYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY
GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL TNLG
APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVSPGIRRLDALISTSLYKKAGYKEASGSGRADALD
DFDLDMIGSDALDDFDLDMIGSDALDDFDLDMLGSDALDDFDL DMLINSRSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSP
FSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVP
VLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQOLLNOGIPVAPHTTEPMINEYPEAITRLVT
GAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRP
LPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGOMDL SHPPPRGHLDELTTT
LESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF .
ATGGCGGCGGCGTGCCOGGAACTGCGTCAGOTGGCGCAGAGCGATGTGTATTGGGATCCGATTGTGAGCATTGAACCG
GATGGCGTGGAAGAAGTGTTTGATCTGACCGTGCCGGGCCCGCATAACTTTGTGGCGAACGATATTATTGCGCATAACT
CTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACC
GTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAA
CCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCC
AAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGG
ACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCA
AAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGT
TGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAA
GGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCA
AGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTA
TTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCAT
GCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACG
GAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCT
TTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAAC
AAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAG
GTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAA
GCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGT
TGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGAT
CAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTC
CCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAG
CTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCECCCGAAGATAATG
AGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAG
TGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCA
GAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGA
AAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATC
GACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTG
ACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGG
ACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGAT
GACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCG
GATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAG
CGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCA
CCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGC
AGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGG
CTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGC
ACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGG
CGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCAC
CGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGT
GGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGC
CTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTC
TATCGCCGATATGGATTTCTCAGCCTTGCTGGGCTCTGGCAGCGGCAGCCGGGATTCCAGGGAAGGGATGTTTTTGCCG
AAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCATTT
CATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAG
US 2019/0345483 A1 Nov. 14 , 2019
25
- continued
CCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCAC
CTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAG
AAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACC
ACACTTGAGTCCATGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCC
TGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGACTGTCCATCTTCGACACATCTCTGTTTTGA
[0118 ] Cell Culture Transfection and Transduction . AAV9-GFP -Cre experiments . C57BL /6 male mice were
[0119 ] All cells were incubated at 37 ° C. and 5 % CO2. used for in vivo electroporation and intramuscular AAV
[0120 ] C2C12 cells were obtained from the American injections .
Tissue Collection Center (ATCC ,Manassas , Va .), and grown [0127 ] All animals were randomly allocated to treatment
in growth media (DMEM + glutaMAX + 10 % FBS ). Cells groups and handled equally .
were split with TypLE Express (Invitrogen ) every 2-3 days [0128 ] In vivo electroporation . Animals were anesthetized
and before reaching 80 % confluency, to prevent terminal using isoflurane and injected with 50 ul of 2 mg/ml
differentiation . Passage number was kept below 15. For hyaluronidase (Sigma- Aldrich , H4272) in the tibialis ante
transfection of C2C12 myoblasts , 10 % cells were plated per riormuscle. After 1 hr,plasmids in vehicle ( 10 mM Tris-HC1
well in a 24 -well plate, in 500 ul of growth media . The pH 8.0 ) were injected into the muscle, followed by elec
following day, fresh media was replaced , and 800 ng of total troporation (Aihara , H. & Miyazaki, J. Gene transfer into
plasmid DNA was transfected with 2.4 ul of Lipofectamine muscle by electroporation in vivo . Nature biotechnology 16 ,
2000 (Life Technologies ). 1: 1 mass ratio of vectors encoding 867-870 , doi: 10.1038 /nbt0998-867 ( 1998 )) ( 10 pulses of 20
Cas9:gRNA (s ) was used . Media was replaced with differ ms at 100 V /cm with 100 ms intervals ) using an ECM 830
entiation media (DMEM + glutaMAX + 2 % donor horse Electro Square Porator (BTX Harvard Apparatus) and a
serum ) on the 1st and 3rd days post-lipofection . two -needle array.
[0121] For differentiation of C2C12 into myotubes, 2x104 [0129 ] For conditions with immunosuppression , FK506
cells were plated per well in a 96 -well plate, in 100 ul of (Sigma- Aldrich , F4679) was administered daily at 5 mg/kg
growth media . At confluency, 1-2 day (s) after plating , media (body weight), commencing 1 day before electroporation .
was replaced with fresh differentiation media (DMEM + [0130 ] For Cas9 -specific T -cell clonotyping and antibody
glutaMAX + 2 % donor horse serum ), and further incubated epitope -mapping, both tibialis anteriormuscles of 11 -week
for 4 days . Fresh differentiation media was replaced before old male C57BL /6 mice were each electroporated with 30 ug
transduction with AAVs. Culture media was replaced with of PSMVP- Cas9F2. Control mice were electroporated with
fresh differentiation media 1d after transduction , and cells 30 ug of plasmid vector control ( consisting of the same
incubated for stated durations. plasmid with Cas9 coding sequence removed ) per muscle.
[0122 ] The 3xStop - tdTomato reporter cell line was Vehicle electroporations were similarly performed . For
derived from tail-tip fibroblasts ofAi9 (Madisen , L. et al. A intramuscular IL -2 and perforin immunofluorescence stain
robust and high - throughput Cre reporting and characteriza ing and centrally nucleated myofiber quantification , 30 ug of
tion system for the whole mouse brain . Nat Neurosci 13 , DNA minicircles vectors encoding SpCas9F2 , 30 ug total of
133-140 , doi:10.1038/ nn.2467 ( 2010 )) mouse ( JAX PCRII -U6 - gRNA , and / or 15 ug of PCAG -GFP were used as
007905 ), and immortalized with lentiviruses encoding the indicated .
large SV40 T - antigen (GenTarget Inc , LVP016 -Puro ). Cells [0131] Genotyping and Analysis.
were cultured in DMEM + pyruvate + glutaMAX + 10 % FBS. [0132 ] C2C12 cells were harvested 4 days post- lipofec
Lipofectamine 2000 (Life Technologies) was used for trans tion , with 100 ul of QuickExtract DNA Extraction Solution
fection of plasmids , and images were taken 5 days after (Epicentre ) per well of a 24 -well plate ; and C2C12 myo
transfection . For transduction with AAVs, cells were plated tubes were harvested 7 days post-AAV transduction , with 20
at 2x104 per well in a 96 -well plate, in 100 ul of growth ul of DNA QuickExtract per well of a 96 -well plate. Cell
media . AAV -containing lysates or purified AAVs were lysates were heated at 65 ° C. for 10 min ., 95 ° C. for 8 min .,
applied at confluency of 70-90 % . Culture media was and stored at -20 ° C. Each locus was amplified from 0.5 ul
replaced with fresh growth media the next day, and cells of cell culture lysate per 25 ul PCR reaction , for 20-25
incubated for stated durations. cycles .
[0123 ] The GC - 1 spg mouse spermatogonial cell line [0133 ] Bulk tissues were each placed in 100 ul of Quick
( CRL - 2053 ) was obtained from ATCC . Cells were cultured Extract DNA Extraction Solution , and heated at 65 ° C. for
and transduced similarly to the 3xStop-tdTomato cell line , 15 min ., 95 ° C. for 10 min . 0.5 ul of lysate was used per 25
with a Cas9N :Cas9C of 1: 1 . ul PCR reaction , and thermocycled for 25 cycles .
(0124] Animals. [0134 ] For barcoding for deep sequencing , 1 ul of each
[0125 ] All animal procedures were approved by the Har unpurified PCR reaction was added to 20 ul of barcoding
vard University Institutional Animal Care and Use Commit PCR reaction , and thermocycled [95 ° C. for 3 min ., and 10
tee . cycles of (95 ° C. for 10 s, 72 ° C. for 65 s)]. Amplicons were
[0126 ] Ai9 (Madisen , L. et al. A robust and high - through pooled , the whole sequencing library purified with self
put Cre reporting and characterization system for the whole made SPRI beads (9 % PEG final concentration ), and
mouse brain . Nat Neurosci 13 , 133-140 , doi:10.1038 /nn . sequenced on a Miseq (Illumina ) for 2x251 cycles. FASTQ
2467 ( 2010 )) mice (JAX No. 007905 ) were used for tdTo were analyzed with BLAT (Kent, W. J. BLAT — the BLAST
mato activation , and for systemic AAV9-Cas9 - gRNAs and like alignment tool. Genome Res 12 , 656-664 , doi: 10.1101/
US 2019/0345483 A1 Nov. 14 , 2019
26
gr.229202 ( 2002)) (with parameters —t= dna q = dna of whole tissues , samples were taken from the heart body
tileSize = 11 -stepSize = 5 oneOff = 1 -rep wall, liver, gastrocnemius muscle, olfactory bulb , ovary,
Match = 10000000 -minMatch = 4 -minIdentity = 90 testis , and diaphragm .
-maxGap 3no= Head ) and post-alignment analyses per [0142 ] AAV9-Cas9 -VPR -gRNAs were intramuscularly
formed with MATLAB (Math Works ). Alignments due to injected at AAV9-Cas9N -ORNAS:AAV9- Cas9C- VPR ratio of
primer dimers were excluded by filtering off sequence 1 :1 , at a total of 4E12 vg . To demarcate transduced tissues,
alignments that did not extend > 2 bp into the loci from the 1E11 of AAV9- turboRFP was coadministered in the same
locus -specific primers . To minimize the impact of sequenc mix . Controls mice were injected with 1E11 of AAV9
ing errors, conservative variant calling was performed by turboRFP only , with the final injection mix at the same
ignoring base substitutions, and calling only variants that volume.
overlap with a 130 bp window from the designated Cas9 [0143] For determining AAV9- and Cas9 - specific T-cell
ORNA cut sites. Negative controls were equally analyzed for clonotypes and antibody epitopes, both tibialis anterior
baseline sequencing error rates, to which statistical tests muscles of 11 -week old male C57BL /6 mice were each
were performed against. injected with AAV9- Cas9N and AAV9- Cas9C (2E12 vg
[0135 ] Off-target sites for Mstn gRNAs were predicted each ). For control mice , 4E12 of AAVO vector control
using the online CRISPR Design Tool(Hsu , P. D. et al. DNA (consisting of the same AAV genome with the Cas9 coding
targeting specificity of RNA - guided Cas9 nucleases. Nature sequence removed ) was injected per muscle. Vehicle
biotechnology 31, 827-832 , doi:10.1038/nbt.2647 (2013 )) (1xPBS + 35 mM NaCl) injections were similarly performed .
(world wide website crispr.mit.edu ). Off -target sites were For IL - 2 and perforin immunofluorescence staining and
ranked by number of mismatches to the on - target sequence , centrally nucleated myofiber quantification, muscles were
and deep sequencing performed on top hits . Sequencing injected with AAV9-Cas9 - VPR - gRNAs at 4E12 vg and
reads were analyzed equally between experimental samples AAV9-turboRFP at 1E11 vg , while control mouse muscles
(AAV9 -Cas9 -gRNASM3 -M4) and control samples (AAV9 were injected with the same volume of vehicle and AAV9
Cas9 -gRNAsTAL+ TdR ) using BLAT. Variant calls were per turboRFP at 1E11 vg.
formed for insertions and deletions that lie within a +15 bp [0144 ] Quantitative PCR (qPCR ) for AAV Genomic Cop
window from potential off - target cut-sites . ies in Tissues.
[0136 ] Quantitative Reverse - Transcription PCR ( ART [0145 ] Each qPCR reaction consists of 1x FastStart Essen
PCR ) for Gene Expression. tial DNA Probes Master (Roche # 06402682001), 100 nM of
[0137 ] Cells were processed with Taqman Cells -to -Ct kits each hydrolysis probe (against the AAV ITR and the mouse
( Thermo Fisher Scientific # 4399002 ) as per manufacturer's Acvr2b locus), 340 nM of AAV ITR reverse primer, 100 nM
instructions, with the modification that each qRT-PCR reac each for all other forward and reverse primers , and 2.5 ul of
tion was scaled down to 25 ul. Taqman hydrolysis probes input tissue lysate . A mastermix was first constituted before
( Thermo Fisher Scientific) used : PD - L1 (Mm00452054_ splitting 22.5 ul into each well, after which tissue lysates
ml), FST (Mm00514982_ml), CD47 (Mm00495011_ml), were added . Thermocycling conditions were : [ 95 ° C. 15
and house -keeping gene Abli (Mm00802029_ml). Gene min .; 40 cycles of (95 ° C. 1 min ., 60 ° C. 1 min .)]. FAM and
expression from targeted genes were normalized to that of HEX fluorescence were taken every cycle. AAV genomic
Abli (Act) and fold -changes were calculated against AAV copies per mouse diploid genome were calculated against
Cas9C - VPR -only controls (no -gRNA ) (2- AAC ). Basal gene standard curves. For each tissue sample, two repeated sam
expression percentiles for C2C12 myotubes and GC - 1 sper plings were performed for qPCR and deep -sequencing , all
matogonial cells ( type B. spermatogonia ) were retrieved on separate days, and the means plotted with s.e.m. qPCR
from the Gene Expression Omnibus (GEO ) repository false positive rate were calculated similarly from two
(GDS2412 and GDS2390 respectively ). vehicle -injected negative control mice, to which statistical
[ 0138 ] Total RNA from skeletal muscle tissues was tests were performed against.
extracted via TRIzol. Reverse transcription was conducted [0146 ] Cas9 Re -Stimulation and TCR -B Repertoire
with High - Capacity cDNA Reverse Transcription Kit (Ap Sequencing
plied Biosystems # 4368814 ), and 5 ul of each reaction was [0147] Lymphocytes were isolated from 2x inguinal
used for qRT-PCR in 1x FastStart Essential DNA Probes lymph nodes and 1x popliteal lymph node per bilaterally
Master (Roche # 06402682001). Gene expression from tar injected mouse . Lymph nodes were scored , and incubated
geted genes were normalized to that of Abli (ACt) and for 30 min . in RPMI+ 1 mg/ml collagenase at RT. Lympho
fold - changes were calculated against AAV9-turboRFP - only cytes were released by meshing through 70 um nylon sieves,
controls (2 -AAC ). washed twice with 1xPBS + 5 mM EDTA , and resuspended
[0139 ] AAV Administration in Mice . in 500 ul of growth media (RPMI 1640 + 10 % FBS + 1xPen /
[0140 ] AAV and control experiments were conducted in a Strep / AmphoB +50 UM 2 -BME ). Cell counting was per
randomized and double -blind fashion . The allocation code formed on a Countess device (Life Technologies). For Cas9
was unblinded only after analyses were completed . AAV9 restimulation experiments , 2.5 ug of recombinant Cas9
Cas9- gRNAs injections utilized AAV9- Cas9N - gRNA : (NEB ) was incubated with > 106 cells in 500 ul of growth
AAV9-Cas9C -P2A - turboGFP ratio of 1: 1. media for 3 days. Non - restimulated wells were conducted in
(0141] 3 -day old neonates were each intraperitoneally parallel without Cas9. Cellular RNA was extracted using
injected with 4E12, 5E11, or 2.5E11 vector genomes (vg ) of QIAshredder and RNeasy micro ( Qiagen ). Reverse tran
total AAV9. Vector volumes were kept at 100 ul. Animals scription was performed with SMARTscribe (Clontech ),
were euthanized via CO2 asphyxia and cervical dislocation using SMARTNNN template -switching adapter as described
3 weeks following injections. For AAV9 -GFP and AAV9 (Mamedov, I. Z. et al.Preparing unbiased T -cell receptor and
mCherry co -transduction experiments , animals were eutha antibody cDNA libraries for the deep next generation
nized 9 days after injection . For qPCR and deep sequencing sequencing profiling . Frontiers in immunology 4 , 456 , doi:
US 2019/0345483 A1 Nov. 14 , 2019
27
10.3389/ fimmu.2013.00456 ( 2013 )) . KAPA HiFi poly eluant was neutralized with 15 ul of 1 M Tris -HCl, pH 8.5 .
merase was used for PCR . Individual RNA molecules were 5 ul of captured phage display eluant was used per PCR
counted based on Unique Molecular Identifiers using reaction , with 20 cycles of spacer amplification and 10
MIGEC ( Shugay , M. et al. Towards error-free profiling of cycles of barcoding, and sequenced on a Miseq ( Illumina ).
immune repertoires. Nature methods 11 , 653-655, doi:10 . Each serum sample was processed for technical replication
1038 /nmeth.2960 (2014 )), aligned with MIXCR (Bolotin , on separate days . Differential binding of phage was deter
D. A. et al. MiXCR : software for comprehensive adaptive mined using DESeq2 (Love , M. I., Huber, W. & Anders, S.
immunity profiling. Nature methods 12 , 380-381, doi: 10 . Moderated estimation of fold change and dispersion for
1038 /nmeth.3364 (2015)), and post-analysis performed with RNA- seq data with DESeq2. Genome biology 15 , 550 ,
MATLAB (MathWorks ). Morisita -Horn indices per expo doi:10.1186 /s13059-014-0550-8 ( 2014 )) in R ( Team , R. C.
sure condition were calculated by pairwise comparisons (ISBN 3-900051-07-0 , 2014 )), with all Cas9-unexposed
among 4 mice ( 2 animals from electroporation dataset and 2 (n = 16 animals ) or AAV- unexposed (n = 16 animals) samples
animals from AAV dataset). as appropriate controls for non -specific binding . Alignments
[0148 ] Fluorescent Immunoassay. and further analyses were performed with MATLAB (Math
[0149] To determine antibody specificity and class -switch Works ). Visualization of epitopes on Cas9 (PBD ID : 4CMP,
ing, serum levels of AAV9- specific IgM , IgG , and IgG2a chain A ) and AAVO VP3 (PBD ID : 3UX1) structures was
from AAV9- treated mice were compared to that from conducted with Pymol. Phenotypic data of double -alanine
vehicle- injected control mice . AAVI viruses (1E9 vg) were AAV9 mutants were obtained from Adachi, K., Enoki, T.,
coated on each well of a 96 -well PVDF MaxiSorp plate for Kawano , Y., Veraz, M. & Nakai, H. Drawing a high
1 hr in 1xTBST, followed by 1 hr of blocking in 1xTBST + resolution functional map of adeno-associated virus capsid
3 % BSA . After 3 washes with 1xTBST, 1 : 100 diluted mouse by massively parallel sequencing. Nat Commun 5 , 3075 ,
serum was applied at 25 ul per well, for 1 hr. After 3 washes doi:10.1038 /ncomms4075 (2014 ), with mutant viral blood
with 1xTBST, 1: 200 diluted anti-mouse secondary antibod persistency calculated as the difference in blood viral levels
ies were added [goat anti-mouse IgG -CF633 (Biotium 72 hr. and 10 min . post- injection (both normalized to that of
20120 ), goat anti -mouse IgG2a -CF594 (Biotium 20259 ) and wildtype AAV9 , with 0 denoting wildtype phenotype , and
goat anti-mouse IgM -Dy550 (Pierce PISA510151 )], and negative values denoting loss of blood persistency ).Mutant
incubated for 1 hr. Wells were then washed 5x with 1xTBST, tropism represented by ' Phenotypic Difference ' values as
and fluorescence readings in 100 ulof 1xTBS were taken via described in Adachi, K., Enoki, T., Kawano, Y., Veraz , M. &
a plate reader. All steps were conducted at RT. To account for Nakai, H. Drawing a high - resolution functional map of
autofluorescence from sera , fluorescence readings were nor adeno -associated virus capsid by massively parallel
malized against that from wells treated similarly except for sequencing. Nat Commun 5 , 3075 , doi: 10.1038 /
the exclusion of secondary antibodies . ncomms4075 (2014 ). Solvent-accessibility surface area
[0150 ] Epitope Mapping by M13 Phage Display. (sasa ) ratios for AAV9 capsid (PDB ID : 3UX1) were first
[0151] M13KE genome was amplified by PCR , with one calculated as described in Mandell, D. J. et al. Biocontain
end terminating with the pIII peptidase cleavage signal , and ment of genetically modified organisms by synthetic protein
the other end terminating with a 4xGly linker followed by design . Nature 518 , 55-60 , doi: 10.1038 /nature14121 (2015 ),
the mature pIII. Cas9 and AAVO VP1 capsid coding and the final sasa ratio per residue calculated as the mean
sequence PCR products were each randomly fragmented from a 15 bp sliding window centered on the residue .
with NEBNext dsDNA Fragmentase until about 50-300 bp . 0152 ] Total mRNA -Sequencing .
Purified fragments were treated with NEBNext End -Repair [0153 ] 1 ug of TRIzol-extracted RNA from muscle tissues
Module . After DNA purification , fragments were blunted were enriched for polyA -tailed mRNA and processed with
ligated into the M13KE PCR product overnight at 16 ° C. NEBNext Ultra Directional RNA Library Prep Kit (New
The entire ligation reaction was purified , and transformed England Biolabs), followed by sequencing on a NextSeq
into ER2738 (Lucigen ), at 200 ng per 25 ul ofbacteria, with sequencer ( Illumina ), giving 30 million reads per sample .
electroporation conditions of 10 uF, 60092 , and 1.8 kV. After Reads were aligned to the mm10 reference genome and
30 min . recovery in SOC media , the culture was amplified FPKM quantified with the Cufflinks workflow ( Trapnell, C.
by combining with 20 mlof early -log ER2738 culture . After et al. Transcript assembly and quantification by RNA -Seq
4 hrs, the culture supernatant was collected , and incubated to reveals unannotated transcripts and isoform switching dur
a final concentration of 3.33 % PEG - 8000 and 417 mM of ing cell differentiation . Nature biotechnology 28 , 511-515 ,
NaCl, overnight at 4 ° C. M13 phage was pelleted , and doi:10.1038 /nbt.1621 ( 2010 )), with differential expression
resuspended in 2 ml of TBS. Phage titers were determined tested with Cuffdiff ( Trapnell , C. et al. Differential analysis
by LB /IPTG /X -gal blue -white plague counting, averaging of gene regulation at transcript resolution with RNA - seq .
> 1E11 pfu /ul. For Ig:phage pull -down, 20 ul of each phage Nature biotechnology 31 , 46-53 , doi: 10.1038 /nbt.2450
library was incubated with 5 ul of mouse serum or titrated (2013)). GO -terms network was visualized with ClueGO
amount of purified antibody controls [7A9 (Novus Bio ), (Bindea, G. et al. ClueGO : a Cytoscape plug - in to decipher
Guide- IT ( Clontech ), bG15 (Santa Cruz ), bS18 (Santa functionally grouped gene ontology and pathway annotation
Cruz ), bD20 (Santa Cruz ), non -binding mouse IgG isotype networks. Bioinformatics 25 , 1091-1093 , doi: 10.1093/bio
control (Santa Cruz)], and made up to 50 ulwith TBST, for informatics/btp101 (2009 )) Cytoscape (Shannon , P. et al .
1 hr at RT. For each reaction , 25 ul of Protein A /G magnetic Cytoscape: a software environment for integrated models of
beads (Millipore PureProteome) was first washed twice with biomolecular interaction networks. Genome Res 13 , 2498
TBST, resuspended to 10 ul, and added to the reaction for 2504 , doi: 10.1101 /gr.1239303 (2003 )) plug - in .
additional 30 min . incubation . The beads were then washed [0154 ] Histology and Immunofluorescence Staining .
5x with TBST, and captured Ig :phage eluted with 100 ul of [0155 ] Mouse organs and tissue samples were dissected ,
200 mM glycine-HC1, pH 2.2 , 1 mg/ml BSA for 8 min . The fixed in 4 % paraformaldehyde in 1xDPBS for 1.5 hr, fol
US 2019/0345483 A1 Nov. 14 , 2019
28
lowed by 3x5 min . washes with 1xDPBS. Samples were GFP and mCherry fluorescence intensities above the back
immersed in 30 % sucrose until submersion, embedded in ground thresholds were identified , and the lower intensity
O.C.T. compound ( Tissue- Tek ), frozen in liquid -nitrogen values from either channel were used to populate a merged
cold isopentane, and cryosectioned on a Microm HM550 image . All other pixels in the merged image were set to null.
( Thermo Scientific ). Skeletal muscles were sectioned to a [0166 ] Whole organ images were taken on a SMZ1500
thickness of 12 um , while the liver and heart were sectioned (Nikon ) fluorescence dissection stereomicroscope equipped
at 20 um . with a SPOT RT3 camera (Diagnostic Instruments), for an
[0156 ] For immunofluorescence , TA muscle sections were imaging area of 16 mm by 12 mm , with 3 s exposure for the
blocked in 1xPBST + 3 % BSA for 1 hr at RT, immunostained liver and 4 s exposure for the heart , muscle, brain and
with primary antibodies at RT for 1 hr, followed by 3x gonads . All images were acquired with a gain setting of 8
washes with PBS/ T . Slides were then incubated with sec using the SPOT imaging software (Diagnostic Instruments ,
ondary antibodies at RT for 1 hr, followed by 3x washes with Sterling Heights , Mich .). Images for each organ were
PBS / T. Anti -mouse IL -2 and perforin antibodies were used inverted and thresholded equally across animals.
at 1: 100 (Santa Cruz sc -7896 and sc -9105 respectively), [0167 ] Images were analyzed with Image ) (NIH ), Cell
followed by 1:200 of secondary anti-rabbit CF633 (Bi Profiler (Carpenter, A. E. et al. CellProfiler: image analysis
otium ). Immunostained slides were mounted with mounting software for identifying and quantifying cell phenotypes .
media containing DAPI (Vector Laboratories, H1500 ). Genome biology 7 , R100 , doi:10.1186 / gb - 2006-7-10 -r100
[0157] Western Blot. (2006 )) and MATLAB (Math Works ).
[0158] Muscles were harvested 2 weeks after AAV injec
tions or plasmid electroporation . -10 mm? tissue clippings Cas9 Orthologs and Applications of AAV -Split-Cas9
were flash - frozen in liquid nitrogen , followed by lysis in
300-500 ul of T-PER Tissue Protein Extraction Solution Targeting Range of Cas9 Orthologs (FIG . 1A )
( Thermo Scientific ) supplemented with 1x Complete Pro [0168 ] Cas9 orthologs , such as those from S. aureus (Sa )
tease Inhibitor (Roche ), and homogenized in gentleMACS (Ran , F. A. et al. In vivo genome editing using Staphylo
M tubes (Miltenyi Biotec ). 10-15 ul of each tissue lysate was coccus aureus Cas9. Nature 520 , 186-191, doi: 10.1038 /
ran on 8 % Bolt Bis - Tris Plus gels (Life Technologies ) in 1x nature14299 ( 2015 )), S. thermophilus (St1) (Esvelt, K.M. et
BoltMOPS SDS running buffer at 165 V for 50 min . Protein al. Orthogonal Cas9 proteins for RNA -guided gene regula
transfer was performed with iBlot (Life Technologies ) onto tion and editing . Nature methods 10 , 1116-1121, doi: 10 .
PVDF membranes , using program 3 for 13 min . Western 1038 /nmeth.2681 (2013 )) and N. meningitides (Nm ) (Esvelt ,
blots were conducted with 1 :200 of anti-Cas9 Guide - IT K. M. et al. Orthogonal Cas9 proteins for RNA -guided gene
polyclonal antibody (Clontech 632607 ), 1 :400 of anti regulation and editing . Nature methods 10 , 1116-1121 , doi:
GAPDH polyclonal antibody (Santa Cruz sc -25778 ), and 10.1038 /nmeth.2681 (2013 )), present exciting and comple
1 :2500 of anti-rabbit IgG -HRP secondary antibody (Santa mentary ways to manipulate the genome. However, with
Cruz sc- 2004 ), using an iBind device (Life Technologies). more restrictive PAM requirements , these orthologs are
Stained membranes were developed with SuperSignal West biologically unable to recognize the large spectrum of
Femto Maximum Sensitivity Substrate ( Thermo Scientific ) genomic sites accessible by SpCas9 . This diminishes the key
and imaged on Chemidoc MP ( Bio -Rad ). Band intensities attractiveness of CRISPR -Cas9 in facile DNA- addressing .
were quantified with ImageJ (NIH ). PAM requirements can be altered by artificially evolving
10159 ] Immunosuppression . Cases, which significantly broadens the targeting ranges
[0160] FK506 was dissolved in 100 % DMSO , and the (Kleinstiver, B. P. et al. Engineered CRISPR - Cas9 nucleases
stock solution further diluted 1: 100 in vehicle for final with altered PAM specificities. Nature 523, 481-485, doi:
concentrations of 1 % DMSO , 10 % Cremophor (Sigma 10.1038 /nature14592 ( 2015 ); Kleinstiver, B. P. et al. Broad
Aldrich , C5135), and 1xPBS. Mice were injected daily with ening the targeting range of Staphylococcus aureus
5 mg/kg (body weight) of FK506 , with the first injection CRISPR -Cas9 by modifying PAM recognition . Nature bio
commencing 1 day before in vivo electroporation . technology , doi:10.1038 /nbt.3404 (2015 )). Considering
[0161] 30 ug of minicircle- SMVP- Cas9 was injected for canonical, non -canonical and altered PAMs, SpCas9
Cas9 -only injections, 15 ug of PCAG -GFP for GFP -only requires an NRG , NGR , or NGCG PAM , while SaCas9
injections, and 30 ug ofminicircle - SMVP -Cas9 and 15 ug of requires an NNGRR or NNNRRT PAM (these are referred
PCAG -GFP for Cas9 +GFP injections. 4 mice were injected to as Sp * and Sa * respectively in FIG . 1A ). Wenote that this
per condition . is an underestimate of the current Sp * Cas9 targeting range ,
[0162 ] Imaging and Analyses . because SpCas9 fused with DNA -binding domains allows
[0163 ] Confocal images were taken using a Zeiss LSM780 targeting of the NGC PAM (Bolukbasi, M. F. et al. DNA
inverted microscope . For live cell-imaging , each image binding-domain fusions enhance the targeting range and
consists of 3x z -stacks (7 um intervals ) and 2x2 tiles. For precision of Cas9. Nature methods 12 , 1150-1156 , doi:10 .
muscle sections, 3x z -stacks (7 um intervals ) were used . For 1038 /nmeth.3624 (2015 )). The relaxed NGC PAM is not
liver and heart sections , 4x z -stacks ( 10 um intervals ) were included in our analysis , in the spirit to maintain conserva
used . For imaging of all tissue sections, tiling to cover entire tive comparison in the absence of such engineering con
samples were used , followed by stitching . Stacked fluores ducted with any of the other Cas9 orthologs . We also note
cence images were projected by maximum intensity with that in the context of AAV delivery , in order to harness the
Zen 2011 (Carl Zeiss ). SpCas9 - fusion variants for enhancing targeting range and
[0164 ] Epifluorescence images were taken with an Axio specificity , a split- Cas9 approach would be necessary .
Observer D1 (Carl Zeiss ) or Axio Observer Z1 (Carl Zeiss ). Together, these engineering efforts further increase the gap
[0165 ] For co - transduction analysis after AAVI-GFP and between Sp * and Sa * Cases against the other orthologs, with
AAV9-mCherry administration , pixels that contain both Sp * Cas9 retaining the broadest targeting range .
US 2019/0345483 A1 Nov. 14 , 2019
29
[0169 ] The advantage of a relaxed PAM is exponential target DNA . Cell 156 , 935-949 , doi: 10.1016 / j.cell.2014.02.
when multiple sites are targeted within a genome, where the 001 (2014 ); Jinek , M. et al. Structures of Cas9 endonu
probability of finding multiple suitable sites is a product of cleases reveal RNA -mediated conformational activation .
the PAM densities. A useful application of multiplex Science 343 , 1247997 , doi: 10.1126 / science.1247997
CRISPR -Cas9 would be to generate genomic excisions, as (2014 ); Nishimasu , H. et al. Crystal Structure of Staphylo
we apply here . The PAM density also dictates the feasibility coccus aureus Cas9. Cell 162, 1113-1126 , doi:10.1016 /j.
ofwidely used CRISPR -Cas9 tools . For example, specificity cell.2015.08.007 (2015 ); Hirano , H. et al. Structure and
of CRISPR - Cas9 gene -targeting is significantly increased Engineering of Francisella novicida Cas9. Cell 164 , 950
with the use of paired Cas9-nickases (Mali, P. et al. CAS9 961 , doi: 10.1016 / j.cell.2016.01.039 (2016 )), it is foreseen
transcriptional activators for target specificity screening and this split -reconstitution engineering framework to be generi
paired nickases for cooperative genome engineering . Nature cally applicable , even for orthologs yet to be characterized ,
biotechnology 31, 833-838, doi:10.1038/nbt.2675 (2013); which would likely be necessary when novel Cas9 and Cpfi
domain fusions are to be made ( such as with more specific
Ran , F. A. et al. Double nicking by RNA - guided CRISPR
Cas9 for enhanced genome editing specificity . Cell 154 , and efficient nucleolytic domains, epigenetic effectors, pro
1380-1389 , doi: 10.1016 /j.cell.2013.08.021 (2013 ) ) or tein complex recruiters , inducible domains, nucleotide base
dCas9 -Foki ( Tsai, S. Q. et al. Dimeric CRISPR RNA- guided editors, and the like ). These protein domains generally range
Foki nucleases for highly specific genome editing. Nature in the hundreds to thousands ofbase -pairs . For comparison,
biotechnology 32 , 569-576 , doi: 10.1038 /nbt.2908 (2014 ); the all -in -one AAV-SpCas9FL -gRNA (Senis, E. et al.
Tsai, S. Q. et al. Dimeric CRISPR RNA - guided Foki nucle CRISPR /Cas9 -mediated genome engineering : an adeno -as
ases for highly specific genome editing. Nature biotechnol sociated viral (AAV ) vector toolbox . Biotechnology journal
ogy 32, 569-576 , doi: 10.1038/nbt.2908 (2014 )), which 9 , 1402-1412 , doi: 10.1002/biot.201400046 (2014 )) and
requires proximal binding of two Cas9 - gRNA complexes to AAV -SaCas9FL -ORNA (Ran , F. A. et al. In vivo genome
effect double - strand breaks. Importantly, these approaches editing using Staphylococcus aureus Cas9. Nature 520 ,
operate on the basis that endonucleolytic activity is consti 186-191, doi: 10.1038 /nature14299 (2015 )) designs as
tuted only when both Cas9 -gRNA complexes are within a described are > 4.8 kb , and would not be able to accommo
certain molecular distance from each other (< 100 bp for date additional elements in their current forms.
offset nicking with Cas9 -nickases; 15 bp or 25 bp for [0173 ] Furthermore, we show here that genome- editing
dCas9 -Foki). Existence of two Cas9 - gRNA target sites frequency is highly dependent on delivery efficiency. The
within these specific distances is hence necessary for func split- reconstitution paradigm could also grant compatibility
tion . The numbers of human (FIG . 1A ) and mouse exonic of Cas9s and Cpfls with self - complementary AAV (payload
sites that can be targeted with these specificity -enhancing limit of 2.4-3.3 kb ), which confers transduction efficiency
approaches are orders of magnitude higher for SpCas9 , often superior to conventional single -stranded AAVs (Mc
compared to the other orthologs. Carty , D. M. Self -complementary AAV vectors; advances
Activity of Cas9 Orthologs
and applications. Mol Ther 16 , 1648-1656 , doi: 10.1038/mt.
2008.171 (2008)).
[0170 ] While SpCas9 has been successfully employed to Split -Cpfl and Split-Ago
target a myriad of genomic sites across a broad spectrum of
species, it is now well -documented that individual gRNAs [0174] On a similar design principle as our approach ,
can exhibit variable targeting efficiencies. Likewise , there splitting Cpf1 orthologs at sites that maximize the likelihood
are hints that the other orthologs exhibit similar variability . for proper folding of each lobe might be attempted. Struc
For example , in the first demonstration of SaCas9 for tures for Cpfl proteins have been determined (Dong D , Ren
gene-editing in the liver (Ran , F. A. et al. In vivo genome K , Qiu X , Zheng J, Guo M , Guan X , Liu H , Li N , Zhang B ,
editing using Staphylococcus aureus Cas9. Nature 520 , Yang D ,Ma C , Wang S , Wu D , Ma Y , Fan S , Wang J, Gao
186-191, doi:10.1038 /nature14299 (2015)), Pcsk9 was tar N , Huang Z , The crystal structure of Cpfi in complex with
geted at -40 % , while Apob at 0 % to 8.9 % . CRISPR RNA , Nature , 2016 April 28 ; 532 (7600 ):522-6 ;
[0171 ] It would hence be illuminating to compare activi Yamano T , Nishimasu H , Zetsche B , Hirano H , Slaymaker
ties of various orthologs on a global scale, to determine if I M , Li Y , Fedorova I, Nakane T, Makarova K S , Koonin E
putative target sites can be edited , and how efficiently if so . V , Ishitani R , Zhang F , Nureki O , Crystal Structure ofCpfi
For example , StiCas9 generally underperformed SpCas9 in Complex with Guide RNA and Target DNA , Cell, 2016
across > 1000 tested gRNAs (Chari, R., Mali, P. , Moos May 5 ; 165(4 ): 949-62). From the LbCpfl structure (PDB
burner, M. & Church , G. M. Unraveling CRISPR - Cas9 ID : 51D6 ), the less structured linkers at V280- E292 , Q513
genome engineering parameters via a library -on -library K520 , and N803- F810 might be appropriate split -sites .
approach . Nature methods 12 , 823-826 , doi: 10.1038/nmeth . From the AsCpfl structure (PDB ID : 5B43 ), the less struc
3473 ( 2015 )). Comprehensive comparisons between tured linkers between S311 - S325 , T522 -K530 ,M795 - E804 ,
CRISPR -Cas9s and CRISPR -Cpfls are anticipated for these and N878 -K887 might be appropriate split-sites. Because
highly enticing systems. both structures were determined from nucleic -acid (s ) bound
AAV -Split - Cas9 Allows Domain Fusions and Compatibility Cpfl proteins, free Cpfl apoenzyme structuresmight reveal
with Self-Complementary AAVS more of such potential split-sites.
[0172] Split -Cas9 shortens the coding sequences signifi [0175 ] Similarly , previously determined protein structures
cantly below all known Cas9 orthologs, which liberates the show that Argonaute proteins are also bilobal, and hence
severely limited AAV capacity for the many exciting appli would be appropriate for the current approach . The long
cations with CRISPR - Cas9. Since Cas9 and Cpf1 proteins prokaryotic argonautes comprise of an N -terminal PAZ lobe
generally adopt a bi- lobed structure (Nishimasu, H. et al. and a C - terminal PIWI lobe, connected by Linker 2 (L2 )
Crystal structure of Cas9 in complex with guide RNA and (See Swarts , D. C. et al. The evolutionary journey of
US 2019/0345483 A1 Nov. 14 , 2019
30
Argonaute proteins. Nature structural & molecular biology Tissue -Specificity and Small-Molecule Regulation of
21, 743-753 , doi: 10.1038 /nsmb.2879 (2014); Song, J. J., AAV -Cas9 - gRNA
Smith , S. K., Hannon , G. J. & Joshua- Tor, L. Crystal [0178 ] In this study, we demonstrate use of an excision
structure of Argonaute and its implications for RISC slicer dependent fluorescence reporter mouse line (Madisen , L. et
activity . Science 305, 1434-1437 , doi: 10.1126 /science . al. A robust and high -throughput Cre reporting and charac
1102514 ( 2004); Wang , Y., Sheng, G., Juranek , S., Tuschl, T. terization system for the whole mouse brain . Nat Neurosci
& Patel, D. J. Structure of the guide-strand -containing 13, 133-140, doi: 10.1038 /nn.2467 (2010 )) for detecting
argonaute silencing complex . Nature 456 , 209-213 , doi: 10 . Cas9 -gRNA activity in situ . Demarcation of AAV9- Cas9
1038 /nature07315 ( 2008); Sheng, G. et al. Structure -based gRNA biodistribution revealed edited cells across multiple
cleavage mechanism of Thermus thermophilus Argonaute tissue types and organs, enabled by the robustness ofAAVO
DNA guide strand-mediated DNA target cleavage. Proceed
ings of the National Academy of Sciences of the United for systemic delivery . On the other hand, this wide viral
States of America 111, 652-657 , doi:10.1073/pnas. spread urges that careful monitoring and confinement of
1321032111 ( 2014 )). This conserved bilobal architecture AAV -Cas9 -gRNA would be prudent. Enticingly , the dual
suggests that the L2 domain is an appropriate split-site. For AAVs format offers multi-tiered safeguards to restrict Cas9
PfAgo (PDB : 1U04 ), the L2 domain lies E276 -R363. Within ORNA activity to specific tissues of interest . The ability to
the L2 , Q347 -L356 is less structured and might be most use independent transcriptional and translational elements
preferred . Based on the PfAgo structure , the less structured within the two AAVs would enable stricter tissue-specific
region at Y413 - E443 might also be an appropriate split-site . regulation , such as by intersecting two or more tissue
For TtAgo (PDB : 3DLH , 4N47), the L2 domain lies E272 specific elements. In addition , using independentAAV sero
F338 . Based on the TtAgo structure , D269-W283 , R315
L321, and T504 -P515 are less structured regions, and might types for each split -half and gRNA (s ) would further confine
be most preferred . NgAgo structure has not been deter Cas9 - gRNA function to tissues where tropisms overlap
mined , but based on the high structural conservation among Enhancing tissue -level specificity complements the
orthologs between species and across prokaryotes to eukary increased genome-level specificity that has been demon
otes, a similar bilobal protein structure is likely. From strated with Cas9 engineering (Mali , P. et al. CAS9 tran
homology alignment of NgAgo with PfAgo and TtAgo scriptional activators for target specificity screening and
using HHPred (Soding , J., Biegert, A. & Lupas, A. N. The paired nickases for cooperative genome engineering . Nature
HHpred interactive server for protein homology detection biotechnology 31, 833-838 , doi: 10.1038 / nbt.2675 (2013);
and structure prediction . Nucleic Acids Res 33, W244-248 , Ran , F. A. et al. Double nicking by RNA - guided CRISPR
doi: 10.1093 /nar/gki408 ( 2005)), Q417 -A438 , Y481- T502, Cas9 for enhanced genome editing specificity . Cell 154 ,
and S696-0707 are potential split -sites.
1380-1389 , doi:10.1016 / j.cell.2013.08.021 (2013 ); Tsai, S.
AAV- Cas9 - gRNA for Homologous Recombination (HR ) Q. et al. Dimeric CRISPR RNA - guided Fokl nucleases for
[0176 ] The significantly increased space granted by AAV highly specific genome editing. Nature biotechnology 32 ,
split -Cas9 and evidence of efficientAAV co -transduction are 569-576 , doi: 10.1038 /nbt.2908 (2014 ); Guilinger, J. P. ,
particularly relevant for applications demanding template Thompson , D. B. & Liu , D. R. Fusion of catalytically
directed HR . To accomplish HR , donor DNA templates have inactive Cas9 to Foki nuclease improves the specificity of
to be co -delivered into the cell with Cas9- gRNA . Even when genome modification . Nature biotechnology 32, 577-582 ,
using the smaller Cas9 orthologs (~ 3.3 kb ), the incorpora doi: 10.1038 /nbt.2909 (2014 ); Slaymaker, I. M. et al. Ratio
tion of viral ITRs (0.3 kb ), generic transcriptional regulators nally engineered Cas9 nucleases with improved specificity.
( -0.8 kb ) and a single polIII promoter- gRNA cassette (0.4 Science , doi: 10.1126 /science.aad5227 ( 2015 )). For
kb ) would already push the payload (~ 4.8 kb ) to the limit of example , intein insertions can render full - length SpCas9
AAV capacity , precluding the incorporation of a HR donor conditionally inactive/active in response to small molecules,
sequence. This implies that to accomplish HR - directed thereby also increasing genomic target-specificity (Davis , K.
genome-editing , employing dual AAVs would be necessary M., Pattanayak , V., Thompson , D. B., Zuris , J. A. & Liu , D.
( Yang, Y. et al . A dual AAV system enables the Cas9 R. Small molecule- triggered Cas9 protein with improved
mediated correction of a metabolic liver disease in newborn
mice . Nature biotechnology , doi: 10.1038 /nbt.3469 (2016 )), genome- editing specificity . Nat Chem Biol 11 , 316-318 ,
in line with the approach undertaken in this study . doi: 10.1038 /nchembio.1793 (2015) ). However , none of the
[0177 ] Secondly, both non -homologous end - joining 15 tested intein - inserted Cas9 variants retained full Cas9FL
(NHEJ) and HR are processes downstream of dsDNA cleav activity , potentially due to disruption of the Cas9 structure ;
age, and editing frequencies via either mechanism would furthermore , the coding sequences of these variants (5.4 kb )
depend on and reflect the extent of Cas9 -mediated dsDNA exceed the AAV payload limitation . To capitalize on the
cleavage . Hence , editing via NHEJ could serve as a first increased targeting specificity of small -molecule inducible
proxy for potential HR . We characterize the first steps in this Cas9, it is foreseen that combining inducible split- inteins
process , showing the direct dependence of NHEJ frequency (Mootz , H. D., Blum , E. S., Tyszkiewicz, A. B. & Muir , T.
with delivery efficiency across organs. This suggests that HR W. Conditional protein splicing : a new tool to control
efficiency would likely also depend on delivery efficiency. protein structure and function in vitro and in vivo . J Am
Subsequent investigations into developmental stage , cell
states and types, and donor sequence properties will be Chem Soc 125 , 10561-10569, doi: 10.1021/ja0362813
necessary to pinpoint and optimize the parameters underly (2003 )) and structure -guided engineered AAV-split -Cas9
ing competence for HR following systemic delivery of ( this study) would confer exquisite functional regulation in
Cas9 -gRNA . vivo .
US 2019/0345483 Al Nov. 14 , 2019
31
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS : 50

< 210 > SEQ ID NO 1
< 211 > LENGTH : 14
< 212 > TYPE : PRT
< 213 > ORGANISM : Artificial Sequence
< 220 > FEATURE :
< 223 > OTHER INFORMATION : Description of Artificial Sequence : Synthetic
peptide
< 400 > SEQUENCE : 1
Cys Ala Ser Ser Leu Asp Arg Gly Gln Asp Thr Gln Tyr Phe
1 5 10
< 210 > SEQ ID NO 2

< 211 > LENGTH : 1368
< 212 > TYPE : PRT
< 213 > ORGANISM : Streptococcus pyogenes
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gin Leu Val Gin Thr Tyr
180 185 190
Asn Gin Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gin Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gin Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gin Ile Gly Asp Gln Tyr Ala Asp
US 2019/0345483 A1 Nov. 14 , 2019
32
- continued
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gin Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gin Gin Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gin Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gin Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu lu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gin Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gin Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gin Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gin Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gin
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gin Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gin Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
US 2019/0345483 A1 Nov. 14 , 2019
33
- continued
Ala Asn Arg Asn Phe Met Gin Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gin Lys Ala Gin Val Ser Gly Gin Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gin Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gin
755 760 765
Thr Thr Gln Lys Gly Gin Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gin Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gin Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gin Leu Leu Asn Ala Lys Leu Ile Thr Gin Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gin Leu Val Glu Thr Arg Gin Ile Thr
915 920 925
Lys His Val Ala Gin Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gin Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gin Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
US 2019/0345483 A1 Nov. 14 , 2019
34
- continued
Arg Lys Val Leu Ser Met Pro Gin Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gin Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gin Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gin Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gin Leu Gly Gly Asp
1355 1360 1365
< 210 > SEQ ID NO 3

< 211 > LENGTH : 83
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt ttt 83
< 210 > SEQ ID NO 4

< 211 > LENGTH : 9
< 212 > TYPE : PRT
US 2019/0345483 A1 Nov. 14 , 2019
35
- continued
Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1 5
< 210 > SEQ ID NO 5

< 211 > LENGTH : 12
< 212 > TYPE : PRT
Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro
1 5 10
< 210 > SEQ ID NO 6

< 211 > LENGTH : 10
< 212 > TYPE : PRT
Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1 5 10
< 210 > SEQ ID NO 7

< 211 > LENGTH : 19
< 212 > TYPE : PRT
< 213 > ORGANISM : Adeno - associated virus
Phe Met Ile Pro Gin Tyr Gly Tyr Leu Thr Leu Asn Asp Gly Ser Gin
1 5 10 15
Ala Val Gly
< 210 > SEQ ID NO 8

< 211 > LENGTH : 10
< 212 > TYPE : PRT

Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
1 5 10
< 210 > SEQ ID NO 9

< 211 > LENGTH : 12
< 212 > TYPE : PRT
Thr Gln Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly
1 5 10
< 210 > SEQ ID NO 10

< 211 > LENGTH : 40
< 212 > TYPE : DNA
< 220 > FEATURE :
< 223 > OTHER INFORMATION : Description of Artificial Sequence: Synthetic
oligonucleotide
< 400 > SEQUENCE : 10
gggccatgtg gacatccatg aggtgagaca gtgccagcgt 40
US 2019/0345483 A1 Nov. 14 , 2019
36
- continued
< 210 > SEQ ID NO 11

< 211 > LENGTH : 38
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 11
ggcctgaagc cactacagct gctggagatc aaggctcg 38
< 210 > SEQ ID NO 12

< 211 > LENGTH : 27
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 12
ggccctagca tctaagttct cgcaggc 27
< 210 > SEQ ID NO 13

< 211 > LENGTH : 35
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 13
ggtcattcca tctcagctgt gacagcagcg cagaa 35
< 210 > SEQ ID NO 14

< 211 > LENGTH : 30
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 14
gtcaagccca aagtctctcc gggacctctt 30
< 210 > SEQ ID NO 15

< 211 > LENGTH : 29
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 15
ggaatcccgg tgctgccgct accccctca 29
< 210 > SEQ ID NO 16

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 16
US 2019/0345483 A1 Nov. 14 , 2019
37
- continued
gctagagaat aggaacttct t 21
< 210 > SEQ ID NO 17

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 17
gaaagaattg atttgatacc g 21
< 210 > SEQ ID NO 18

< 211 > LENGTH : 19
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 18
gatccccato aagctgato 19
< 210 > SEQ ID NO 19

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 19
ggtatgctat acgaagttat t 21
< 210 > SEQ ID NO 20

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 20
gctcgagata agacc 15
< 210 > SEQ ID NO 21

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 21
gctaaagtca tccgc 15
< 210 > SEQ ID NO 22

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
US 2019/0345483 A1 Nov. 14 , 2019
38
- continued
< 400 > SEQUENCE : 22

ggttcttatt tgcgt 15
< 210 > SEQ ID NO 23

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 23
ggaaatcaaa gcggc 15
< 210 > SEQ ID NO 24

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 24
gaaggagttc ctcgg 15
< 210 > SEQ ID NO 25

< 211 > LENGTH : 15
< 212 > TYPE : DNA
< 220 > FEATURE :
oligonucleotide
< 400 > SEQUENCE : 25
gaggaggtcc acttc 15
< 210 > SEQ ID NO 26

< 211 > LENGTH : 54
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 220 > FEATURE :
< 221 > NAME / KEY : modified_base
< 222 > LOCATION : (29 ) .. ( 34 )
< 223 > OTHER INFORMATION : a , C , t , g , unknown or other
< 400 > SEQUENCE : 26
ctttccctac acgacgctct tccgatctnn nnnnctggag tgttagagtg ggcg 54
< 210 > SEQ ID NO 27

< 211 > LENGTH : 48
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 27
ggagttcaga cgtgtgctct tccgatctga ctgccccatg gaaagaca 00
< 210 > SEQ ID NO 28

US 2019/0345483 A1 Nov. 14 , 2019
39
- continued
< 211 > LENGTH : 58
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 220 > FEATURE :
< 221 > NAME /KEY : modified_base
< 222 > LOCATION : ( 29 ) .. ( 34 )
< 223 > OTHER INFORMATION : ? , C , t , g , unknown or other
< 400 > SEQUENCE : 28
ctttccctac acgacgctct tccgatctnn nnnngggcca tgaaaggaaa aatgaagt 58
< 210 > SEQ ID NO 29

< 211 > LENGTH : 48
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 29
ggagttcaga cgtgtgctct tccgatctgc ctctggggtt tgcttggt 48
< 210 > SEQ ID NO 30

< 211 > LENGTH : 67
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 220 > FEATURE :
< 221 > NAME /KEY : modified_base
< 222 > LOCATION : ( 29 ) .. ( 34 )
< 223 > OTHER INFORMATION : ? , C , t , g , unknown or other
< 400 > SEQUENCE : 30
ctttccctac acgacgctct tccgatctnn nnnngagata taagctgaat aaggccaatg 60
acatact 67
< 210 > SEQ ID NO 31

< 211 > LENGTH : 48
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 31
ggagttcaga cgtgtgctct tccgatctct actgctcttt cctgccga 48
< 210 > SEQ ID NO 32

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 32
gcctactcgc tgctgcccat t 21
< 210 > SEQ ID NO 33

< 211 > LENGTH : 21
US 2019/0345483 A1 Nov. 14 , 2019
40
- continued
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 33
cctggagacc cccaaaagct C 21
< 210 > SEQ ID NO 34

< 211 > LENGTH : 19
< 212 > TYPE : DNA
< 220 > FEATURE :
probe
< 400 > SEQUENCE : 34
agatcttccc acttcaggt 19
< 210 > SEQ ID NO 35

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 35
ggaaccccta gtgatggagt t 21
< 210 > SEQ ID NO 36

< 211 > LENGTH : 16
< 212 > TYPE : DNA
< 220 > FEATURE :
primer
< 400 > SEQUENCE : 36
cggcctcagt gagcga 16
< 210 > SEQ ID NO 37

< 211 > LENGTH : 21
< 212 > TYPE : DNA
< 220 > FEATURE :
probe
< 400 > SEQUENCE : 37
cactccctct ctgcgcgctc g 21
< 210 > SEQ ID NO 38

< 211 > LENGTH : 831
< 212 > TYPE : PRT
< 220 > FEATURE :
polypeptide
< 400 > SEQUENCE : 38
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
US 2019/0345483 A1 Nov. 14 , 2019
41
- continued
Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
20 25 30
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
35 40 45
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
50 55 60
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
65 70 75 80
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
85 90 95
Tyr Leu Gin Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
100 105 110
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
115 120 125
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
130 135 140
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
145 150 155 160
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
165 170 175
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
180 185 190
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gin Leu Val Gin Thr Tyr
195 200 205
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
210 215 220
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
225 230 235 240
Leu Ile Ala Gin Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
245 250 255
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
260 265 270
Asp Leu Ala Glu Asp Ala Lys Leu Gin Leu Ser Lys Asp Thr Tyr Asp
275 280 285
Asp Asp Leu Asp Asn Leu Leu Ala Gin Ile Gly Asp Gin Tyr Ala Asp
290 295 300
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
305 310 315 320
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
325 330 335
Met Ile Lys Arg Tyr Asp Glu His His Gin Asp Leu Thr Leu Leu Lys
340 345 350
Ala Leu Val Arg Gin Gin Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
355 360 365
Asp Gin Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
370 375 380
Gin Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
385 390 395 400
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
405 410 415
Lys Gin Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gin Ile His Leu
US 2019/0345483 A1 Nov. 14 , 2019
42
- continued
420 425 430
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
435 440 445
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
450 455 460
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
465 470 475 480
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
485 490 495
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
500 505 510
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
515 520 525
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
530 535 540
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
545 550 555 560
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
565 570 575
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
580 585 590
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
595 600 605
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
610 615 620
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
625 630 635 640
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
645 650 655
His Leu Phe Asp Asp Lys Val Met Lys Gin Leu Lys Arg Arg Arg Tyr
660 665 670
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
675 680 685
Lys Gin Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
690 695 700
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
705 710 715 720
Lys Glu Asp Ile Gin Lys Ala Gin Val Cys Leu Ala Gly Asp Thr Leu
725 730 735
Ile Thr Leu Ala Asp Gly Arg Arg Val Pro Ile Arg Glu Leu Val Ser
740 745 750
Gin Gin Asn Phe Ser Val Trp Ala Leu Asn Pro Gin Thr Tyr Arg Leu
755 760 765
Glu Arg Ala Arg Val Ser Arg Ala Phe Cys Thr Gly Ile Lys Pro Val
770 775 780
Tyr Arg Leu Thr Thr Arg Leu Gly Arg Ser Ile Arg Ala Thr Ala Asn
785 790 795 800
His Arg Phe Leu Thr Pro Gln Gly Trp Lys Arg Val Asp Glu Leu Gln
805 810 815
Pro Gly Asp Tyr Leu Ala Leu Pro Arg Arg Ile Pro Thr Ala Ser
820 825 830
US 2019/0345483 A1 Nov. 14 , 2019
43
- continued
< 210 > SEQ ID NO 39

< 211 > LENGTH : 2496
< 212 > TYPE : DNA
< 220 > FEATURE :
polynucleotide
< 400 > SEQUENCE : 39
atggccccaa agaagaagcg gaaggtcggt atccacggag toccagcago cgacaagaag 60
tactccattg ggctcgatat cggcacaaac agcgtcggct gggccgtcat tacggacgag 120
tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag 180
aagaacctca ttggcgccct cctgttcgac tccggggaaa cggccgaagc cacgcggctc 240
aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag 300

atctttagta atgagatggc taaggtggat gactctttct tccataggct ggaggagtcc 360
tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa tatcgtggac 420
gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa gottgtagac 480
agtactgata aggctgactt gcggttgato tatctcgcgc tggcgcatat gatcaaattt 540
cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt cgacaaactc 600
tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat caacgcatcc 660
ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg gctcgaaaac 720
ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct tatcgccctg 780
tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga tgccaagctt 840
caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca gatcggcgac 900
cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct gctgagtgat 960
attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat gatcaagcgc 1020
tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca gcaactgcct 1080
gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg atacattgac 1140
ggcggagcaa gccaggagga attttacaaatttattaagc ccatcttgga aaaaatggac 1200
ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa acagcgcact 1260

ttcgacaatg gaagcatccc ccaccagatt cacctgggcg aactgcacgc tatcctcagg 1320
cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga gaaaatcctc 1380
acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg 1440
atgactcgca aatcagaaga gaccatcact ccctggaact tcgaggaagt cgtggataag 1500
ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa tctgcctaac 1560
gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta taacgagcto 1620
accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc tggagagcag 1680
aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt gaaacagctc 1740
aaagaagact atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag cggagtggag 1800
gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat taaagacaag 1860
gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct cacccttacg 1920

US 2019/0345483 A1 Nov. 14 , 2019
44
- continued
ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca tctcttcgac 1980
gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg gctgtcaaga 2040

aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga ttttcttaag 2100
tccgatggat ttgccaaccg gaacttcatg cagttgatcc atgatgactc tctcaccttt 2160
aaggaggaca tccagaaagc acaagtttgt ctggctggcg atactctcat taccctggcc 2220
gatggacgac gagtgcctat tagagaactg gtgtcacagc agaatttttc cgtgtgggct 2280
ctgaatcctc agacttaccg cctggagagg gctagagtga gtagagcttt ctgtaccggc 2340
atcaaacctg tgtaccgcct caccactaga ctggggagat ccattagggc cactgccaac 2400
caccgatttc tcacacctca gggctggaaa cgagtcgatg aactccagcc tggagattac 2460
ctggctctgc ctaggagaat ccctactgcc tcctga 2496
< 210 > SEQ ID NO 40

< 211 > LENGTH : 970
< 212 > TYPE : PRT
< 220 > FEATURE :
polypeptide
< 400 > SEQUENCE : 40
Met Ala Ala Ala Cys Pro Glu Leu Arg Gln Leu Ala Gln Ser Asp Val
1 5 10 15
Tyr Trp Asp Pro Ile Val Ser Ile Glu Pro Asp Gly Val Glu Glu Val
20 25 30
Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val Ala Asn Asp Ile
35 40 45
Ile Ala His Asn Ser Gly Gin Gly Asp Ser Leu His Glu His Ile Ala
50 55 60
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gin Thr Val
65 70 75 80
Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu
85 90 95
Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gin Thr Thr Gln Lys Gly
100 105 110
Gin Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys
115 120 125
Glu Leu Gly Ser Gin Ile Leu Lys Glu His Pro Val Glu Asn Thr Gin
130 135 140
Leu Gin Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gin Asn Gly Arg Asp
145 150 155 160
Met Tyr Val Asp Gin Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
165 170 175
Val Asp His Ile Val Pro Gin Ser Phe Leu Lys Asp Asp Ser Ile Asp
180 185 190
Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
195 200 205
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gin
210 215 220
Leu Leu Asn Ala Lys Leu Ile Thr in Arg Lys Phe Asp Asn Leu Thr
225 230 235 240
US 2019/0345483 Al Nov. 14 , 2019
45
- continued
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile
245 250 255
Lys Arg Gin Leu Val Glu Thr Arg Gin Ile Thr Lys His Val Ala Gin
260 265 270
Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu
275 280 285
Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
290 295 300
Phe Arg Lys Asp Phe Gin Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr
305 310 315 320
His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu
325 330 335
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr
340 345 350
Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gin Glu Ile
355 360 365
Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
370 375 380
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro
385 390 395 400
Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
405 410 415
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gin Val Asn
420 425 430
Ile Val Lys Lys Thr Glu Val Gin Thr Gly Gly Phe Ser Lys Glu Ser
435 440 445
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp
450 455 460
Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
465 470 475 480
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
485 490 495
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
500 505 510
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
515 520 525
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
530 535 540
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gin
545 550 555 560
Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr
565 570 575
Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu
580 585 590
Gin Lys Gln Leu Phe Val Glu Gin His Lys His Tyr Leu Asp Glu Ile
595 600 605
Ile Glu Gin Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
610 615 620
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro
625 630 635 640
Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
US 2019/0345483 A1 Nov. 14 , 2019
46
- continued
645 650 655
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg
660 665 670
Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
675 680 685
Gin Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gin Leu
690 695 700
Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Ser Arg
705 710 715 720
Ala Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp
725 730 735
Val Glu Glu Asn Pro Gly Pro Met Pro Ala Met Lys Ile Glu Cys Arg
740 745 750
Ile Thr Gly Thr Leu Asn Gly Val Glu Phe Glu Leu Val Gly Gly Gly
755 760 765
Glu Gly Thr Pro Glu Gin Gly Arg Met Thr Asn Lys Met Lys Ser Thr
770 775 780
Lys Gly Ala Leu Thr Phe Ser Pro Tyr Leu Leu Ser His Val Met Gly
785 790 795 800
Tyr Gly Phe Tyr His Phe Gly Thr Tyr Pro Ser Gly Tyr Glu Asn Pro
805 810 815
Phe Leu His Ala Ile Asn Asn Gly Gly Tyr Thr Asn Thr Arg Ile Glu
820 825 830
Lys Tyr Glu Asp Gly Gly Val Leu His Val Ser Phe Ser Tyr Arg Tyr
835 840 845
Glu Ala Gly Arg Val Ile Gly Asp Phe Lys Val Val Gly Thr Gly Phe
850 855 860
Pro Glu Asp Ser Val Ile Phe Thr Asp Lys Ile Ile Arg Ser Asn Ala
865 870 875 880
Thr Val Glu His Leu His Pro Met Gly Asp Asn Val Leu Val Gly Ser
885 890 895
Phe Ala Arg Thr Phe Ser Leu Arg Asp Gly Gly Tyr Tyr Ser Phe Val
900 905 910
Val Asp Ser His Met His Phe Lys Ser Ala Ile His Pro Ser Ile Leu
915 920 925
Gin Asn Gly Gly Pro Met Phe Ala Phe Arg Arg Val Glu Glu Leu His
930 935 940
Ser Asn Thr Glu Leu Gly Ile Val Glu Tyr Gln His Ala Phe Lys Thr
945 950 955 960
Pro Ile Ala Phe Ala Arg Ser Arg Ala Arg
965 970
< 210 > SEQ ID NO 41

< 211 > LENGTH : 2913
< 212 > TYPE : DNA
< 220 > FEATURE :
polynucleotide
< 400 > SEQUENCE : 41
atggcggcgg cgtgcccgga actgcgtcag ctggcgcaga gcgatgtgta ttgggatccg 60
attgtgagca ttgaaccgga tggcgtggaa gaagtgtttg atctgaccgt gccgggcccg 120

US 2019/0345483 A1 Nov. 14 , 2019
47
- continued
cataactttg tggcgaacga tattattgcg cataactctg gccaggggga cagtcttcac 180

gagcacatcg ctaatcttgc aggtagocca gctatcaaaa agggaatact gcagaccgtt 240
aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 300
gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 360
aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 420
gaaaacacoc agcttcagaa tgaga agctc tacctgtact acctgcagaa cggcagggac 480
atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 540
gtgccccagt cttttctcaa agatgattet attgataata aagtgttgac aagatccgat 600
aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 660
tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 720

aaggctgaac gaggtggcct gtctgagttg gataaagccg gottcatcaa aaggcagett 780
gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacaco 840
aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 900
ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 960
caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 1020
cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 1080
atcgcaaagt ctgagcagga aataggcaag gocaccgcta agtacttctt ttacagcaat 1140
attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 1200
cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 1260
acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 1320
accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 1380
cgcaaaaaag attgggaccccaagaaatac ggcggattcg attctcctac agtcgcttac 1440
agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 1500
gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 1560

ctcgaggega aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 1620
tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 1680

aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 1740
tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 1800
cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgato 1860
ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 1920
atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 1980
gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 2040
gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 2100
ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa ggtgtctcga 2160
gctggatccg gagccacgaa cttctctctg ttaaagcaag caggggacgt ggaagaaaac 2220
cccggtccta Iccco cat gaaga ag tgccg ca ccggca ct gaacggcgtg 2280
gagttegagc tggtgggcgg cggagagggc acccccgagc agggccgcat gaccaacaag 2340
atgaagagca ccaaaggcgc cctgaccttc agcccctacc tgctgagcca cgtgatgggc 2400

US 2019/0345483 A1 Nov. 14 , 2019
48
- continued
tacggcttct accacttcgg cacctacccc agcggctacg agaacccctt cctgcacgcc 2460

atcaacaacg goggctacac caacacccgc atcgagaagt acgaggacgg cggcgtgctg 2520
cacgtgagct tcagctaccg ctacgaggcc ggccgcgtga tcggcgactt caaggtggtg 2580
ggcaccggct tccccgagga cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc 2640
accgtggagc acctgcaccc catgggcgat aacgtgctgg tgggcagctt cgcccgcacc 2700
ttcagcctgc gcgacggcgg ctactacagc ttcgtggtgg acagecacat gcacttcaag 2760

agcgccatcc accccagcat cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg 2820
gaggagctgc acagcaacac cgagctgggc atcgtggagt accagcacgc cttcaagacc 2880
cccatcgcct tcgccagatc tcgagctcga tga 2913
< 210 > SEQ ID NO 42

< 211 > LENGTH : 1271
< 212 > TYPE : PRT
< 220 > FEATURE :
polypeptide
< 400 > SEQUENCE : 42
Met Ala Ala Ala Cys Pro Glu Leu Arg Gin Leu Ala Gin Ser Asp Val
1 5 10 15
Tyr Trp Asp Pro Ile Val Ser Ile Glu Pro Asp Gly Val Glu Glu Val
20 25 30
Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val Ala Asn Asp Ile
35 40 45
Ile Ala His Asn Ser Gly Gin Gly Asp Ser Leu His Glu His Ile Ala
50 55 60
Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gin Thr Val
65 70 75 80
Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu
85 90 95
Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gin Thr Thr Gln Lys Gly
100 105 110
Gin Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys
115 120 125
Glu Leu Gly Ser Gin Ile Leu Lys Glu His Pro Val Glu Asn Thr Gin
130 135 140
Leu Gin Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gin Asn Gly Arg Asp
145 150 155 160
Met Tyr Val Asp Gin Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
165 170 175
Val Asp His Ile Val Pro Gin Ser Phe Leu Lys Asp Asp Ser Ile Asp
180 185 190
Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
195 200 205
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gin
210 215 220
Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
225 230 235 240
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile
US 2019/0345483 A1 Nov. 14 , 2019
49
- continued
245 250 255
Lys Arg Gin Leu Val Glu Thr Arg Gin Ile Thr Lys His Val Ala Gin
260 265 270
Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu
275 280 285
Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp
290 295 300
Phe Arg Lys Asp Phe Gin Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr
305 310 315 320
His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu
325 330 335
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr
340 345 350
Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile
355 360 365
Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
370 375 380
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro
385 390 395 400
Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
405 410 415
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gin Val Asn
420 425 430
Ile Val Lys Lys Thr Glu Val Gin Thr Gly Gly Phe Ser Lys Glu Ser
435 440 445
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp
450 455 460
Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
465 470 475 480
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu
485 490 495
Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
500 505 510
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
515 520 525
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu
530 535 540
Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gin
545 550 555 560
Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr
565 570 575
Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu
580 585 590
Gin Lys Gin Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile
595 600 605
Ile Glu Gin Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
610 615 620
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro
625 630 635 640
Ile Arg Glu Gin Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
645 650 655
US 2019/0345483 A1 Nov. 14 , 2019
50
- continued
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg
660 665 670
Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
675 680 685
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gin Leu
690 695 700
Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Ser Pro
705 710 715 720
Gly Ile Arg Arg Leu Asp Ala Leu Ile Ser Thr Ser Leu Tyr Lys Lys
725 730 735
Ala Gly Tyr Lys Glu Ala Ser Gly Ser Gly Arg Ala Asp Ala Leu Asp
740 745 750
Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp
755 760 765
Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met
770 775 780
Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Ile Asn
785 790 795 800
Ser Arg Ser Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Gin
805 810 815
Tyr Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys
820 825 830
Arg Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe Ser
835 840 845
Gly Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile Ala Val Pro Ser
850 855 860
Arg Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gin Pro Tyr Pro Phe
865 870 875 880
Thr Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr Met Val
885 890 895
Phe Pro Ser Gly Gin Ile Ser Gin Ala Ser Ala Leu Ala Pro Ala Pro
900 905 910
Pro Gin Val Leu Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro Ala Met
915 920 925
Val Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro Val Leu Ala Pro
930 935 940
Gly Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys Pro Thr Gin Ala
945 950 955 960
Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu Gin Leu Gln Phe Asp Asp
965 970 975
Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val Phe
980 985 990
Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn
995 1000 1005
Gln Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro Met Leu Met
1010 1015 1020
Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala Gln Arg
1025 1030 1035
Pro Pro Asp Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu Pro
1040 1045 1050
US 2019/0345483 A1 Nov. 14 , 2019
51
- continued
Asn Gly Leu Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp
1055 1060 1065
Met Asp Phe Ser Ala Leu Leu Gly Ser Gly Ser Gly Ser Arg Asp
1070 1075 1080
Ser Arg Glu Gly Met Phe Leu Pro Lys Pro Glu Ala Gly Ser Ala
1085 1090 1095
Ile Ser Asp Val Phe Glu Gly Arg Glu Val Cys Gln Pro Lys Arg
1100 1105 1110
Ile Arg Pro Phe His Pro Pro Gly Ser Pro Trp Ala Asn Arg Pro
1115 1120 1125
Leu Pro Ala Ser Leu Ala Pro Thr Pro Thr Gly Pro Val His Glu
1130 1135 1140
Pro Val Gly Ser Leu Thr Pro Ala Pro Val Pro Gin Pro Leu Asp
1145 1150 1155
Pro Ala Pro Ala Val Thr Pro Glu Ala Ser His Leu Leu Glu Asp
1160 1165 1170
Pro Asp Glu Glu Thr Ser Gln Ala Val Lys Ala Leu Arg Glu Met
1175 1180 1185
Ala Asp Thr Val Ile Pro Gln Lys Glu Glu Ala Ala Ile Cys Gly
1190 1195 1200
Gin Met Asp Leu Ser His Pro Pro Pro Arg Gly His Leu Asp Glu
1205 1210 1215
Leu Thr Thr Thr Leu Glu Ser Met Thr Glu Asp Leu Asn Leu Asp
1220 1225 1230
Ser Pro Leu Thr Pro Glu Leu Asn Glu Ile Leu Asp Thr Phe Leu
1235 1240 1245
Asn Asp Glu Cys Leu Leu His Ala Met His Ile Ser Thr Gly Leu
1250 1255 1260
Ser Ile Phe Asp Thr Ser Leu Phe

1265 1270
< 210 > SEQ ID NO 43

< 211 > LENGTH : 3816
< 212 > TYPE : DNA
< 220 > FEATURE :
polynucleotide
< 400 > SEQUENCE : 43
atggcggcgg cgtgcccgga actgcgtcag ctggcgcaga gcgatgtgta ttgggatccg 60
attgtgagca ttgaaccgga tggcgtggaa gaagtgtttg atctgaccgt gccgggcccg 120
cataactttg tggcgaacga tattattgcg cataactctg gccaggggga cagtcttcac 180
gagcacatcg ctaatcttgc aggtagocca gctatcaaaa agggaatact gcagaccgtt 240
aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 300
gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 360
aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 420
gaaaacacoc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 480
atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 540
gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 600

US 2019/0345483 A1 Nov. 14 , 2019
52
- continued
aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 660
tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 720
aaggctgaac gaggtggcct gtctgagttg gataaagccg gottcatcaa aaggcagott 780
gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 840
aagtacgatg aaaatgacaa actgattega gaggtgaaag ttattactct gaagtctaag 900
ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 960
caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 1020
cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 1080
atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 1140
attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 1200
cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 1260
acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 1320
accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 1380
cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 1440
agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 1500

gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 1560
ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 1620
tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 1680
aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 1740
tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 1800
cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 1860
ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 1920
atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 1980
gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 2040
gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 2100
ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa ggtgtcgcca 2160
gggatccgtc gacttgacgc gttgatatca acaagtttgt acaaaaaagc aggctacaaa 2220
gaggccagcg gttccggacg ggctgacgca ttggacgatt ttgatctgga tatgctggga 2280
agtgacgccc tcgatgattt tgaccttgac atgottggtt cggatgccct tgatgacttt 2340
gacctcgaca tgctcggcag tgacgccctt gatgatttcg acctggacat gctgattaac 2400

tctagaagtt coggatctcc gaaaaagaaa cgcaaagttg gtagccagta cctgcccgac 2460
accgacgacc ggcaccggat cgaggaaaag cggaagcgga cctacgagac attcaagagc 2520
atcatgaaga agtccccctt cagcggcccc accgacccta gacctccacc tagaagaatc 2580
gccgtgccca gcagatccag cgccagcgtg ccaaaacctg ccccccagcc ttaccccttc 2640
accagcagcc tgagcaccat caactacgac gagttcccta ccatggtgtt ccccagcggc 2700
cagatctctc aggcctctgc tctggctcca gcccctcctc aggtgctgcc tcaggctcct 2760
gctcctgcac cagctccagc catggtgtct gcactggctc aggcaccagc acccgtgcct 2820
gtgctggctc ctggacctcc acaggctgtg gctccaccag cccctaaacc tacacaggcc 2880

US 2019/0345483 A1 Nov. 14 , 2019
53
- continued
ggcgagggca cactgtctga agctctgctg cagctgcagt tcgacgacga ggatctggga 2940
gccctgctgg gaaacagcac cgatcctgcc gtgttcaccg acctggccag cgtggacaac 3000
agcgagttcc agcagctgct gaaccagggc atccctgtgg cccctcacac caccgagccc 3060
atgctgatgg aataccccga ggccatcacc cggctcgtga caggcgctca gaggcctcct 3120
gatccagctc ctgcccctct gggagcacca ggcctgccta atggactgct gtctggcgac 3180
gaggacttca gctctatcgc cgatatggat ttctcagcct tgctgggctc tggcagcggo 3240
agccgggatt ccagggaagg gatgtttttg ccgaagcctg aggccggctc cgctattagt 3300
gacgtgtttg agggccgcga ggtgtgccag ccaaaacgaa tccggccatt tcatcctcca 3360
ggaagtccat gggccaaccg cccactcccc gccagcctcg caccaacacc aaccggtcca 3420
gtacatgagc cagtcgggtc actgaccccg gcaccagtcc ctcagccact ggatccagcg 3480

cccgcagtga ctcccgaggc cagtcacctg ttggaggatc ccgatgaaga gacgagccag 3540
gctgtcaaag cccttcggga gatggccgat actgtgattc cccagaagga agaggctgca 3600
atctgtggcc aaatggacct ttcccatccg cccccaaggg gccatctgga tgagctgaca 3660
accacacttg agtccatgac cgaggatctg aacctggact cacccctgac cccggaattg 3720
aacgagattc tggatacctt cctgaacgac gagtgcctct tgcatgccat gcatatcago 3780
acaggactgt ccatcttcga cacatctctg ttttga 3816
< 210 > SEQ ID NO 44

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 213 > ORGANISM : Mus sp .
< 400 > SEQUENCE : 44
aagtctctcc gggacctctt ggg 23
< 210 > SEQ ID NO 45

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 400 > SEQUENCE : 45
aaggctctcc aggacctctt ggg 23
< 210 > SEQ ID NO 46

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 400 > SEQUENCE : 46
aagtatctct gggacctctt cag 23
< 210 > SEQ ID NO 47

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 400 > SEQUENCE : 47

gagtccctcc gggagctctt ggg 23
< 210 > SEQ ID NO 48

< 211 > LENGTH : 23
< 212 > TYPE : DNA
US 2019/0345483 A1 Nov. 14 , 2019
54
- continued
< 400 > SEQUENCE : 48
gtgctgccgc taccccctca cgg 23
< 210 > SEQ ID NO 49

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 400 > SEQUENCE : 49
gtgctgcago toccacctca ggg 23
< 210 > SEQ ID NO 50

< 211 > LENGTH : 23
< 212 > TYPE : DNA
< 400 > SEQUENCE : 50
gtgcagccgc taccaccaca aag 23
1. A method of altering a target nucleic acid in a cell 5. The method of claim 1 , wherein the first nucleic acid
comprising and the second nucleic acid are delivered to the cell by
providing to the cell a first nucleic acid encoding a first separate vectors .
portion of a Cas9 protein and a guide RNA (GRNA), 6. The method of claim 1 , wherein the vector is adeno
associated virus.
providing to the cell a second nucleic acid encoding a 7. ( canceled )
second portion of the Cas9 protein and optionally a
transcriptional regulator, 8. ( canceled )
wherein the cell expresses the first portion of the Cas9 9. The method of claim 1 , wherein the first nucleic acid
protein , the gRNA and the second portion of the Cas9 encodes a first portion of the Cas9 protein having first
protein or the second portion of the Cas9 and the split- intein and wherein the second nucleic acid encodes a
transcriptional regulator fusion protein , second portion of the Cas9 protein having a second split
wherein the first portion of the Cas9 protein and the intein complementary to the first split-intein and wherein the
first portion of the Cas9 protein and the second portion of the
second portion of the Cas9 protein , or the first portion Cas9 protein are joined together to form the Cas9 protein .
of the Cas9 protein and the second portion of the Cas9 10. (canceled )
and the transcriptional regulator fusion protein are 11. The method of claim 1, wherein the first portion of the
joined together to form the Cas9 protein or the Cas9 Cas9 protein is the N -terminal lobe of the Cas9 protein and
fusion protein , and the second portion of the Cas9 protein is the C -terminal lobe
wherein the gRNA and the Cas9 protein , or the gRNA and of the Cas9 protein .
the Cas9 fusion protein form a co - localization complex 12.- 15 . ( canceled )
with the target nucleic acid and alter the expression of 16. Themethod of claim 1 ,wherein the Cas9 protein is an
the target nucleic acid . enzymatically active Cas9 protein or a Cas9 protein nickase .
2. The method of claim 1, wherein the first nucleic acid 17. A method of altering a target nucleic acid in a cell of
encodes a first portion of the Cas9 protein having a N -split a subject comprising
intein RmaInts and wherein the second nucleic acid delivering to the cell of the subject a first nucleic acid
encodes a second portion of the Cas9 protein having a encoding a first portion of a Cas9 protein and a guide
C -split -intein RmaIntC and wherein the first portion of the RNA (GRNA) wherein the first nucleic acid is within a
Cas9 protein and the second portion of the Cas9 protein are first vector ,
joined together to form the Cas9 protein . delivering to the cell of the subject a second nucleic acid
3. The method of claim 1 , wherein the first portion of the encoding a second portion of the Cas9 protein and
Cas9 protein is the N -terminal lobe of the Cas9 protein up optionally a transcriptional regulator wherein the sec
to amino acid V713 and the second portion of the Cas9 ond nucleic acid is within a second vector,
protein is the C -terminal lobe of the Cas9 protein beginning wherein the cell expresses the first portion of the Cas9
at D714 . protein , the gRNA and the second portion of the Cas9
4. The method of claim 3, wherein the gRNA having a protein or the second portion of the Cas9 and the
truncated spacer sequence guides the Cas9 protein or the transcriptional regulator fusion protein ,
Cas9 fusion protein to the target nucleic acid and regulate wherein the first portion of the Cas9 protein and the
the expression of the target nucleic acid without cleaving the second portion of the Cas9 protein , or the first portion
target nucleic acid . of the Cas9 protein and the second portion of the Cas9
US 2019/0345483 A1 Nov. 14 , 2019
55
and the transcriptional regulator fusion protein are RmaIntC and wherein the first portion of the Cas9 protein
joined together to form the Cas9 protein or the Cas9 and the second portion of the Cas9 protein are joined
fusion protein , and together to form the Cas9 protein .
wherein the gRNA and the Cas9 protein , or the gRNA and 38.-42 . (canceled )
the Cas9 fusion protein form a co -localization complex 43. The method of claim 35 , wherein the N -terminal
with the target nucleic acid and alter the expression of portion of the Cas9 protein (Cas9M ) is the N - terminallobe of
the target nucleic acid . the Cas9 protein up to amino acid V713 and the C - terminal
18.- 34 . (canceled ) portion of the Cas9 protein is the C -terminal lobe of the Cas9
35. A method ofmodulating a target gene expression in a protein beginning at D714 .
cell comprising providing to the cell a first recombinant 44. (canceled )
adeno - associated virus comprising a first nucleic acid encod 45. The method of claim 35 , wherein the gRNA has
ing an N - terminal portion of the Cas9 protein (Cas9M ) and truncated spacer sequence and directs Cas9FL- TR fusion
a gRNA, protein binding to target DNA without cleaving the target
providing to the cell a second recombinant adeno-asso DNA .
ciated virus comprising a second nucleic acid encoding 46. A method of imaging a target nucleic acid in a cell
a fusion protein comprising a C -terminal portion of the comprising
Cas9 protein (Cas99) fused with a transcriptional regu providing to the cell a first recombinant adeno -associated
lator (TR ), virus comprising a first nucleic acid encoding an N - ter
wherein the cell expresses the Cas9 protein and the minal portion of the Cas9 protein (Cas9M ) and a gRNA ,
Cas9C- TR fusion protein and joins them to form a full providing to the cell a second recombinant adeno -asso
length Cas9tL - TR fusion protein , and ciated virus comprising a second nucleic acid encoding
wherein the cell expresses the gRNA , and the gRNA a fusion protein comprising a C - terminal portion of the
directs the Cas9FL - TR fusion protein to the target gene Cas9 protein (Cas99) fused with a fluorescent protein ,
and modulates target gene expression . wherein the cell expresses the Cas9N protein and the
36. The method of claim 35 , wherein the Cas9 is a Type Cas9 ° fluorescent fusion protein and joins them to form
II CRISPR system Cas9 and the transcriptional regulator is a full length Cas9FL fluorescent fusion protein , and
VPR .
37. The method of claim 35 , wherein the first nucleic acid wherein the cell expresses the gRNA, and the gRNA
encodes the N - terminal portion of the Cas9 protein (Cas97) directs the Cas9FL fluorescent fusion protein to the
having a N - split -intein RmaIntN and wherein the second target nucleic acid and produces fluorescent imaging of
the get nucleic acid .
nucleic acid encodes the fusion protein comprising a C - ter 47.- 75 . (canceled )
minal portion of the Cas9 protein (Cas99 fused with a
transcriptional regulator (TR ) and having a C -split - intein

Crispr

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Crispr

Uploaded by

Copyright:

Available Formats

IND US 20190345483A1

Split-Cas9 AAV Transduction

CAAVRNA lysates (ul/well)

CAG 3x Stop td Tomato C.as9Fb

1E0 1E1 1E2 1E3

AAV9-Cas9C- VPR (2E12 )

AAV9- Cas9N - ORNAFS' (1E12 )

chr 16 : +3906202 AAGGCTCTCCAGGACCTCTT GGG

Mstn on -target M4 GTGCTGCCGCTACCCCCICA CGG 0 2 4

Brain 2.4 vg /dg

Testis < 1 vg/dg

1E0 151 152 1E3

AAV9-Cas9 -gRNASTEL + TOR

AAV9-Cas9 -gRNAS$ 3***

AAV9 -Cas9-9RNAS 1034TR

M-Horisrtna isnmdlaerixty 0.04

Clontypic a(%)bundance 0.6

FIG . 10A Perforin

FIG . 10B Perforin

dysgalactiae equisimilis GGS 124 ; Streptococcus equi - continued

Mstn M3 GTCAAGCCCAAAGTCTCTCCGGGACCTCTT 1 and 2

Ai9 Td5 GCTAGAGAATAGGAACTTCTT

FST F2 GGAAATCAAAGCGGC 1 and 2

TABLE 3 TABLE 3 - continued

Coding sequences of split - Cas9 .

< 160 > NUMBER OF SEQ ID NOS : 50

< 210 > SEQ ID NO 2

< 210 > SEQ ID NO 3

ggcaccgagt cggtgctttt ttt 83

< 210 > SEQ ID NO 4

< 210 > SEQ ID NO 5

< 210 > SEQ ID NO 6

< 210 > SEQ ID NO 7

Ala Val Gly

< 210 > SEQ ID NO 8

< 400 > SEQUENCE : 8

< 210 > SEQ ID NO 9

< 210 > SEQ ID NO 10

< 210 > SEQ ID NO 11

< 210 > SEQ ID NO 12

< 210 > SEQ ID NO 13

< 210 > SEQ ID NO 14

< 210 > SEQ ID NO 15

< 210 > SEQ ID NO 16

< 210 > SEQ ID NO 17

< 210 > SEQ ID NO 18

< 210 > SEQ ID NO 19

< 210 > SEQ ID NO 20

< 210 > SEQ ID NO 21

< 210 > SEQ ID NO 22

< 400 > SEQUENCE : 22

< 210 > SEQ ID NO 23

< 210 > SEQ ID NO 24

< 210 > SEQ ID NO 25

< 210 > SEQ ID NO 26

< 210 > SEQ ID NO 27

< 210 > SEQ ID NO 28

< 210 > SEQ ID NO 29

< 210 > SEQ ID NO 30

< 210 > SEQ ID NO 31

< 210 > SEQ ID NO 32

< 210 > SEQ ID NO 33

< 210 > SEQ ID NO 34