CRISPR/Cas9

Targeting and Inactivation of Viral DNA

Genomes.
Bryan R. Cullen
Center for Virology
Duke University Medical Center

Chronic viral infections cause substantial morbidity and mortality globally,

and represent a serious economic burden.
Hepatitis B Virus
Hepadnavirus.
RNA/DNA hybrid genome w/ small episomal cccDNA intermediate.
In those individuals with chronic infection, frequent complications include hepatocellular
carcinoma and cirrhosis.
Suppressed but not eliminated by antiviral therapy.
300+ Million Globally (Perz et-al 2006).
Human Papilloma Virus
Papilloma Virus.
Small dsDNA episome, occasional aberrant integration results in malignancies.
Head and Neck Cancer: ~21K annually (Gillison 2014).
Cervical Cancer: ~530K new cases in 2008 (Vacarella et-al 2013).
Anal Cancer: ~6K annually

Targeting Integrated HPV-18 in Cervical Carcinoma Cells with

CRISPR/Cas9

Targeting HPV18 with CRISPR/Cas Systems: HPV lifecycle and integration.

These oncogenes are ideal targets for

disruption with Cas9/sgRNA cleavage!

http://www.genpathdiagnostics.com/wp-c ontent/themes/genpa th/images/wh-

gencerv-hpv-full.png

In invasive cervical cancer, HPV

aberrantly integrates (serotype 16
or 18), and these integrants
typically lack E2.
In the absence of E2, E6 and E7
are constitutively expressed and
thus deplete p53 and RB from the
cell.
Without these master cell cycle
regulators the cell proliferates
aggressively, resulting in
transformation.

Integrated HPV18 Inactivation with CRISPR/Cas9 : sgRNA Screening

In F rame Target

HIV-1 Rev

eGFP

Rev

E7 sgRNA 2

E7 sgRNA 1

No sgRNA

Mock

E6 sgRNA 2

E6 sgRNA 1

No sgRNA

Flag

Mock

sgRNA Target

Integrated HPV-18 Inactivation with CRISPR/Cas9 : Cleavage of integrated HPV in HeLa cells.
48hrs

Mock

48hrs

24hrs

Mock

Bp

24hrs

E7 sgRNA 1

E6 sgRNA 1

1,000
500
250
HPV-18 E6 sgRNA 1
5-TACCATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACG-3
3-ATGGTACCGCGCGAAACTCCTAGGTTGTGCCGCTGGGATGTTCGATGGACTAGACACGTGC-5
WT CACCACAATACCATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATC-AACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATC----------------CAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGC----------------GGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCCA------------------CTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGA------AACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCCA-CACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATC--ACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCC-ACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCCA----GGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCCA------------------------GATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAAGATC-------GCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATC-----CGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA
CACCACAATACCATGGCGCGCTTTGAGGATCCA---------CCCTACAAGCTACCTGATCTGTGCACGGAACTGA
A
CACCACAATACCATGGCGCGCTTTGAGGATCCA ACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGA

181x

4x
4x

34x

HPV18 E7 sgRNA 1
5-ACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGAAATAGATGGAGT-3
3-TGGAAGATACAGTGCTCGTTAATTCGCTGAGTCTCCTTCTTTTGCTACTTTATCTACCTCA-5

Wt TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACTCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCAT 180x
TCCGGTTGACCTTCTATGTCACG--------------TCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCAT
TCCGGTTGACCTTCTATGTCACG------------------------AAAACGATGAAATAGATGGAGTTAATCAT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT-----------AACGATGAAATAGATGGAGTTAATCAT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGC---TCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCAT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT---------AAAACGATGAAATAGATGGAGTTAATCAT
TCCGGTTGACCTTCTAT--------------------TCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATCAT
T
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT

CAGAGGAAGAAAACGATGAAATAGATGGAGTTAATC
39x
GT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT
CAGAGGAAGAAAACGATGAAATAGATGGAGTTAATC
AT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT
CAGAGGAAGAAAACGATGAAATAGATGGAGTTAATC 3x
CT
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACT
CAGAGGAAGAAAACGATGAAATAGATGGAGTTAATC 2x
Large Insertion from Human Chr. 8
TCCGGTTGACCTTCTATGTCACGAGCAATTAAGCGACTATACAGTGCAGCTACAGTCTGTGATAAAGCCATGGACACCT
CGTTCCGAGCATTCCAAATGTGTTTAATTGTATCTTTTGGCTTTCATATTTCTTTTTTCTAGGACGATAGGTCTGTTTT
AGTTACATATTTTTTTGCCTGAAGTCAGATCTCACAGCTTTCAGAGGAAGAAAACGATGAAATAGATGGAGTTAATC

Integrated HPV18 Inactivation with CRISPR/Cas9 : E6 and E7 play an essential roll in cellular
transformation.

http://www.intechopen.com/source/html/30755/media/image2.png

E7 sgRNA 1

E6 sgRNA 1

Vector

C
Mut E6t1
WT E6t1

Flag

Act

Flag

Flag
p53

1 0.9 0.5 0.5

Rb

Act
1 1.1 1.1 1.4

Act

+ E6 Mutant

E6 sgRNA 1

E7 sgRNA 1

p21

d
E6 sgRNA 1

p53

5-CG CGC TTT GAG GAC CCC ACG CGG-3

5-CG CGC TTT GAG GAT CCA ACA CGG-3
R F E D P T R

N.S.sgRNA

Mock

N.S. sgRNA

Integrated HPV-18 Inactivation with CRISPR/Cas9 : E6 and E7 disruption induces p53 and RB.

G1
S
G2

10

12.7

15.8

10

10

50

72.5
101

68

400

Gated
hela E6 E7_E6t1.fcs
Event Count: 3371

600
PE-A

1000

NS sgRNA

104

200

400

Gated
hela E6 E7_E7t2.fcs
Event Count: 2844

600
PE-A

800

1000

E6 sgRNA 1 E7 sgRNA 1

104

103

104

31 16 13

10

10

31.4

102

APC-A

APC-A

BrdU-APC

800

E6

200

15.8

10

10

12.7

APC-A

100

sg
R

100

sg
R
N
A

5.67

5.77

103

sg
R
N
A

E7

10

100

104

rel % cells

104

APC-A

APC-A

150

53 9.3
73
5 .7
6 8 5.7

72.5

101

53

10

101

9.27

100
0

200

Gated
hela E6 E7_NS.fcs
Event Count: 5112

400

600
PE-A

800

68

5.77

5.67

1000

10

100
0

200

400

600
PE-A

800

Propidium Iodide

Gated
hela E6 E7_E6t1.fcs

1000

200

400

600
PE-A

800

1000

Brigid Poling and Anand Kornepati

Gated
hela E6 E7_E7t2.fcs

In conjunction with Michael Goldstein and Anand Kornepati

Summary
Cleavage and inactivation of of the integrated HPV-18 E6 and E7 genes in HeLa cells
results in recapitulation of proven phenotypes, which results in highly effective cell
killing.
This proof of concept experiment demonstrates that Spy Cas9 mediated disruption of
viral targets required for transformation is an effective strategy to eliminate cancer
cells

Next Steps
Similar approaches can be applied to other HPV malignancies, including anal and
head and neck cancer, and other viral malignancies, such as EBV-induced
nasopharyngeal carcinoma.
Continue optimization of small Type IIa Cas9 proteins to continue this work in
animals using AAV vectors.

Developing A Minimal Type II CRISPR/Cas9 System for Use in AAV

Vectors

AAV delivery is the optimal modality for delivery of Spy Cas9 effectors to target tissues
AAV has a strict packaging limit, ~4.8kb, including the ITRs.
Spy Cas9 is too large at 4.2kb
Utilize a small Type II Cas9 ortholog from Staph. aureus.

ITR

Gln

BsmBI 2x

143 nt
70nt

U6

BBSI 2X

Int.

EFS

SauCas9 NLS FLAG

251 nt

249 nt
sgRNA 1

sgRNA 2

96 nt

96 nt

3,234 nt

143 nt
Synthetic Poly ( A) site

120nt

Replace U6 (250bp) with a tRNA promoter.

ITR

~60 bp
Optimized minimal synthetic Poly A

Total AAV= 4,462

How can we reduce the size of this vector to permit multiplexing?

Acknowledgements
Cullen Lab
Anand Kornepati
Adam Mefferd
Hal Bogerd
Adam Whisnant
Brigid Poling
Joy Marshall

Schinazi Lab
Leda Basset
Kasten Lab
Michael Goldstein