Chapter

Chapter
THE APPLICATION OF CRISPR/CAS9

THERAPIES IN OPHTHALMOLOGY AND
RECENT ADVANCES FOR THE TREATMENT
OF GENETIC EYE DISEASE
C. B. Tara Moore1, , PhD, Larry A. DeDionisio2, , PhD,

 †
Hila Roshanravan2, , PhD, Connie Chao-Shern2,

‡ §
and M. Andrew Nesbit1, , PhD ‖
1
Ulster University, Coleraine, County Londonderry,
UK/Northern Ireland
2
Avellino Lab USA, Menlo Park, CA, US
 
Author Email: tara.moore@ulster.ac.uk.
† †
Author Email: larry@avellinolabs.com.
‡ ‡
Author Email: hila.roshanravan@avellinolabs.com.
§ §
Author Email: connie@avellinolabs.com.
‖ ‖
Author Email: a.nesbit@ulster.ac.uk.
ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)

and CRISPR-associated (Cas) protein systems have been used to target
chosen regions of the genome with high efficiency and specificity and,
together with the multiplexing capabilities of this technology, have
introduced a novel tool to the field of genome engineering. With a
potential for the permanent treatment of genetic ocular diseases, CRISPR
based therapeutics hold great potential for the field of ophthalmology.
CRISPR systems can repair genetic mutations in recessive diseases where
both copies of a gene carry a pathogenic mutation, or they can knock out
the pathogenic gene allele in dominant diseases where only one copy of
the gene carries the mutation leaving the functionality of the second gene
copy in place. The majority of corneal dystrophies are the result an
autosomal dominant inheritance pattern within the TGFBI gene, which
presents an ideal model suited for a CRISPR/Cas knock out methodology.
This approach can also be applied to the treatment of autosomal
dominantly inherited genetic disease in general. There are several ocular
diseases that are currently being investigated worldwide for which human
gene transfer trials have been approved. This review summarises the use
of CRISPR-Cas gene editing in ophthalmology and focuses on the
advancement of gene editing in the cornea.
1. INTRODUCTION
Vision is often considered the most important human sense. Our eyes
facilitate daily activities from basic mobility to the appreciation of art and
nature. Genetically heritable eye disease have the potential to impact those
who suffer from them in profound ways. In ophthalmology, disease-
causing genetic mutations that lead to retinal disease and the various types
of corneal dystrophy (CD) often display directly observable ocular
manifestations to the clinician. However, the considerable heterogeneity of
inherited ocular disease presents difficulty in choosing a treatment plan.
Many of the inherited, non-syndromic eye diseases are caused by the very
specific spatial expression of the mutated genes (Tucker, Mullins, and
Stone 2014). To date, more than 3000 genetic mutations in approximately
70 genes have been associated with retinitis pigmentosa (RP) (Dias et al.
3 The Application of CRISPR/Cas9 Therapies in Ophthalmology …
2018) and at least 8 genes have been identified for diverse corneal CDs
(Klintworth 2009, Schorderet 2015). The International Committee of the
Classification of Corneal Dytrophies (IC3D) outlined genetic links to 22
different types of CD (Weiss et al. 2015). A precision or personalised
medicine that functions to identify the genetic basis of a disease in the
patient and treat it with a tailored approach is the most effective way to
treat these types of maladies.
Gene therapy is defined as either the introduction of exogenous DNA
or RNA into a patient's cells or the silencing of pathogenic gene mutations
within the patient’s DNA. Silencing endogenous gene mutations known as
gene editing is achieved either by gene knockout or mutation correction.
Genetics based therapeutic approaches include RNA interference (RNAi)
with short interfering RNA (siRNA), gene replacement and gene editing
via programmable nucleases. Gene replacement therapies, where a
functional copy of the gene to treat loss of function mutations commonly
introduced via viral transgene expression have translated to clinical trials
for inherited ocular disease of the retina. These therapies treat autosomal
recessive conditions (Parker et al. 2016, Weleber et al. 2016, Hauswirth et
al. 2008, Bainbridge et al. 2008, Bennett et al. 2016, Ghazi et al. 2016,
Edwards et al. 2016, Michalakis et al. 2017, Dalkara et al. 2016). In this
context, biallelic pathogenic mutations in a disease-associated gene could
be supplemented with the wild type, functional version of the gene.
However, this strategy cannot be applied to dominant conditions, where the
pathogenic gene needs to be repaired or down-regulated. The Moore
research group at Ulster University has extensively studied the application
of siRNA for treatment of autosomal dominantly inherited genetic diseases
of the skin and eye and have clearly demonstrated the potential of
therapeutic siRNA for the cornea (Courtney, Atkinson, Allen, Moore,
Walsh, Pedrioli, MacEwen, Pellegrini, Maurizi, Serafini, et al. 2014,
Courtney, Atkinson, Moore, et al. 2014, Allen et al. 2013, Liao et al. 2011,
McLean and Moore 2011, Atkinson et al. 2011, Moore et al. 2011).
For clinical treatment of autosomal dominant diseases, gene editing
holds enormous potential. Gene editing makes use of programmable
nucleases to target DNA in a sequence specific manner inducing a double
4 C. B. Tara Moore, Larry A. DeDionisio, Hila Roshanravan et al.
strand break (DSB). The DSB allows the mutation within the DNA
sequence to either be removed by non-homologous end-joining (NHEJ) or
repaired via homology directed repair (HDR) (Rudin, Sugarman, and
Haber 1989, Plessis et al. 1992, Rouet, Smih, and Jasin 1994, Choulika et
al. 1995, Bibikova et al. 2001, Bibikova et al. 2003, Bibikova et al. 2002).
Four classes of programmable nucleases are currently used,
meganucleases, (Belfort and Bonocora 2014, Stoddard 2011) zinc finger
nucleases (ZFNs), (Urnov et al. 2010) transcription activator-like effector
nucleases (TALENs) (Bogdanove and Voytas 2011) and clustered
regularly interspersed palindromic repeats (CRISPR) associated nuclease,
Cas9 (Jinek et al. 2012, Cong et al. 2013, Mali, Yang, et al. 2013, Zetsche
et al. 2015). CRISPR/Cas systems are derived from an endogenous defence
mechanism identified within a diverse group of bacteria and archaea
(Karginov and Hannon 2010, Makarova et al. 2011, Makarova et al. 2015).
Compared with the extensive protein engineering required for the
meganucleases and ZFNs, CRISPR/Cas9 can be easily programmed with
simple molecular cloning techniques. Modifications have been engineered
to enhance the CRISPR/Cas9 complex for genome editing within
mammalian cells facilitating the use of CRISPR/CAS9 to treat various
human diseases (Xu et al. 2015, Schwank et al. 2013, Lee et al. 2015).
The ease of using the CRISPR system has resulted in it rapidly
replacing other programmable nucleases as the preferred tool for genome
editing. Gene editing with CRISPR/Cas9 also largely circumvents the limit
of viral vector capacity faced by gene-replacement therapies (Ran et al.
2015, Zuris et al. 2015). This efficient technology is particularly attractive
in ophthalmology. The eye’s accessible anatomical position, immune
privilege and the blood-retinal barrier make it an ideal site for in vivo gene
editing (Liu, Tuo, and Chan 2011). It is foreseen that CRISPR/Cas based
therapy for ocular disease will pave the way for personalised medicine.
This review discusses the progress in the use of CRISPR/Cas systems for
treating eye disease with an emphasis on recent developments in the
treatment of rare and inherited disorders of the cornea.
2. APPROACHES FOR GENE EDITING WITH CRISPR/CAS
Figure 1. Cas9 programmed by tracrRNA:crRNA duplex.
Cas9 is a crucial component in the type II CRISPR/Cas system and

cleaves double-stranded DNA (dsDNA) in a sequence directed and specific
manner. The first step in this protein-DNA interaction is binding of the
Cas9 nuclease to the DNA target site via a single guide (sg) RNA sequence
that is complementary to the target DNA and to a short segment of DNA
adjacent to the target region referred to as the protospacer adjacent motif
(PAM). The PAM acts as a recognition site for Cas9 and is critical in its
ability to cleave the target DNA (Figure 1). Alteration of the 20 nucleotide
spacer sequence within the sgRNA, lying directly upstream of the PAM
directs the RNA-guided endonuclease to the desired target sequence (Jinek
et al. 2012, Xu et al. 2014). Different PAMs are recognised by Cas
nucleases from different bacterial species. The most characterised type II
systems are S. pyogenes Cas9, which recognises a 5–NGG–3′ PAM and S.
aureus Cas9, which recognises a 5–NNGRRT– 3’ PAM (Belfort and

Bonocora 2014, Mojica et al. 2009). The PAM-proximal region of the
guide, often referred to as the ‘seed region’ (Semenova et al. 2011) dictates
the binding of Cas9 to the target site so mismatches in this region are
critical in determining specificity while mismatches in the distal region
have less impact on the specificity (Pattanayak et al. 2013, Sternberg et al.
2014, Wu et al. 2014). There is no uniformity in the exact length of the
seed region; however, the studies suggest it is the first 5-12bp of the guide
sequence that is critical.
2.1. Minimising off Target Cleavage and

Improving Efficiency
There are limitations that can hinder applications of the CRISPR/Cas9

system such as inefficient delivery, dysregulation of the delivered gene, the
immune response against the CRISPR system, and off-target effects or the
unintended on-target mutations at sites that share identical or highly similar
sequence to the target locus (Cho et al. 2014, Fu et al. 2013, Hsu et al.
2013, Pattanayak et al. 2013, Lin, Cradick, et al. 2014, Lee and Kim 2019).
There are studies that determined the mutation frequency in CRISPR/Cas9
edited mouse and monkey models were not significantly higher than in
wild type control animals (Willi et al. 2018, Luo et al. 2018). These studies
did not rule out the possibility of rare mutations at genomic loci that
possess sequence homogeneity to the target sequence. To counteract these
unwanted genomic editing events, the sequence characteristics of the
sgRNA, which are critical for improving specificity and efficiency should
be carefully monitored. Guides with high cleavage efficiency are necessary
within a clinical setting in order to achieve therapeutic benefits. Several
online tools have been designed to allow in silico analyses of on- and off-
target cleavage of each guide sequence. Studies that utilized optimal guide
design in conjunction with genome-wide analyses have reported minimal
off target cleavage (Lin, Cradick, et al. 2014, Kim et al. 2015, Veres et al.
2014, Frock et al. 2015, Tsai et al. 2017).
Table 1. Different techniques to identify off-targets
Methods Description
linear amplification–mediated High coverage sequencing to assess the degree of mutagenesis
high-throughput genome-wide across the entire genome (Hu et al. 2016)
sequencing (LAM-HTGTS)
Di-genome seq in vitro Cas9-digested whole-genome sequencing (Kim et al.
2015)
Circularization for in vitro A highly sensitive, sequencing-efficient in vitro screening
reporting of cleavage effects by strategy for identifying CRISPR-Cas9 genome-wide off-target
sequencing (CIRCLE-seq) mutations (Tsai et al. 2017)
Breaks Labelling, Enrichment The first method for direct labelling of DSBs in situ followed
on Streptavidin, and Sequencing by their genome-wide mapping at nucleotide resolution
(BLESS) (Crosetto et al. 2013)
Breaks Labelling In Situ and 1) Direct labelling of DSBs in fixed cells or tissue
Sequencing (BLISS) sections on a solid surface
2) low-input requirement by linear amplification of
tagged DSBs by in vitro transcription
3) quantification of DSBs through unique molecular
identifiers
4) easy scalability and multiplexing (Yan et al. 2017)
Genome-wide Unbiased Relies on capture of double-stranded oligodeoxynucleotides
Identification of DSBs Enabled into breaks (Tsai et al. 2015)
by Sequencing (GUIDE-Seq)
SITE-Seq A biochemical method, based on the selective enrichment and
identification of adapter-tagged DNA ends by sequencing
(Cameron et al. 2017)
Several methods have been devised to directly identify cleaved sites in

cells (Table 1) and these techniques have achieved good sensitivity and
resolution. However, at the present time there is no single “gold standard
method” which can accomplish unbiased, comprehensive off-target
analysis. To reduce the off-target activity of CRISPR/Cas, much focus has
been directed on improving target specificity of the system. Several
strategies to enhance Cas9 specificity have been reported (Table 2).
In addition to the work carried out to reduce off-target effects, much
effort has been done to enhance the gene editing efficiency of the
CRISPR/Cas9 complex. The DSBs introduced by the enzyme are most
commonly repaired by the NHEJ pathway, leading to nonspecific
nucleotide insertions and deletions, referred to as ‘indels’. This is

convenient for generating gene knockouts but does not allow introduction
of specific sequence changes. The alternative path to repair, HDR enables
a recombination event between a homologous donor DNA template and
damaged DNA resulting in an accurate repair of the DSB. Many groups
have shown different strategies to improve the HDR rate of repair over the
NHEJ pathway (Zhang et al. 2017, Paquet et al. 2016, Kwart et al. 2017,
Liang et al. 2017). For example, the inhibition of DNA ligase IV, the key
enzyme utilized by NHEJ with the inhibitor Scr7 can be used to promote
HDR (Maruyama et al. 2015, Chu et al. 2015, Singh, Schimenti, and
Bolcun-Filas 2015). HDR is restricted to the G2 phase of cell division
when DNA replication is completed and sister chromatids are available to
serve as repair templates, (Lin, Staahl, et al. 2014) which limits the use of
this pathways in certain cell types such as ocular cell types where the cells
are primarily in the post-mitotic G0 state (Young 1985).
Table 2. Different strategies to enhance Cas9 specificity
Techniques Description
reducing the amount of Studies have shown increased off-target activities with high levels of
active Cas9 in cells Cas9 protein(Fu et al. 2013, Hsu et al. 2013, Pattanayak et al. 2013)
and a number of techniques have been employed to restrict the
concentration of Cas9 protein such as delivering it as a purified
protein loaded with sgRNA (Cho et al. 2013, Kim et al. 2014) or
limiting the duration and expression of Cas9, using various inducible
platforms (Davis et al. 2015, Zetsche, Volz, and Zhang 2015).
Double nicking by Cas9 Creates a pair of juxtaposed single-stranded DNA nicks to enhance
nickase mutants genome editing specificity (Ran et al. 2013, Mali, Aach, et al. 2013).
Altering the sgRNA Several studies showed that truncation of sgRNA leads to an
sequence increased target specificity and reduced off target activity.
(Pattanayak et al. 2013, Fu, Reyon, and Joung 2014, Fu et al. 2014)
However, it has been shown by our group that a range of truncated
guide lengths or addition of 5′-GG to the 20 nt guide sequence did
not provide a marked improvement of specificity (Figure 2)
(Christie et al. 2017).
Fusion of catalytically Target DNA sites modified in human cells with >140-fold higher
inactive Cas9 to FokI specificity than wild-type Cas9 and the specificity of fCas9 was
nuclease (fCas9) around 4-fold higher than that of paired nickases at loci with
highly similar off-target sites (Guilinger, Thompson, and Liu 2014,

Tsai et al. 2014). More recent developments have incorporated the
Fokl-Cas9 system with truncated sgRNA to further improve
specificity and reduce off-target activities (Wyvekens et al. 2015).
Techniques Description
Although each of these approaches reduce off-target mutagenesis,
they have a number of limitations: reducing the amount of Cas9 can
decrease on-target cleavage efficiency and truncated guides can
increase indel formation at some off-target loci.
improve the specificity of A study reported that neutralization of positive charges in the non-
Streptococcus pyogenes target groove can dramatically decrease off-target indel formation
Cas9 (SpCas9) is based while preserving on-target activity (Slaymaker et al. 2016). In
on structure-guided
another report, engineering of additional high-fidelity variants of
protein engineering.
CRISPR-associated nucleases by introducing substitutions at
additional non-specific DNA contacting residues can further reduce
some of the very small number of residual off-target sites that persist
for certain sgRNAs (Kleinstiver et al. 2016).
2.2. Expanding the CRISPR/Cas Protospacer

Adjacent Motif (PAM) Repertoire
Cas9 nucleases from various bacterial species have been shown to

have different PAM sequences (Table 3) including a newly characterised
member of the CRISPR programmable nuclease system, Cpf1 from the
Acidaminococcus and Lachnospiraceae bacteria, which was adapted for
genome editing in human cells (Zetsche et al. 2015). Its PAM motif, TTTN
was shown to provide the best cleavage efficiency. The expanding
CRISPR/Cas PAM repertoire will allow greater targeting across diverse
genomic regions. Moreover, it has been reported that artificially
engineered SpCas9 and SaCas9 with alternative PAM recognition motifs
can be generated (Kleinstiver, Prew, Tsai, Nguyen, et al. 2015, Kleinstiver,
Prew, Tsai, Topkar, et al. 2015). Thus, the application of directed evolution
technology to the development of Cas9 with varying PAM motifs will
expand the repertoire of targetable sequences throughout the human
genome and increase the range of accessible therapeutic targets.
Table 3. CRISPR/Cas Protospacer Adjacent Motif (PAM) repertoire
Source Nuclease type PAM sequence

Streptococcus pyogenes Cas9 NGG(Jinek et al. 2012)
Cas9 (D1135E) with improved NGG with (Kleinstiver, Prew, Tsai,
recognition and speficity
Topkar, et al. 2015)
Cas9 (“VQR”; NGAN or NGNG (Kleinstiver,
D1135V/R1335Q/T1337R)
Prew, Tsai, Topkar, et al. 2015)
Cas9 (“EQR”; NGAG (Kleinstiver, Prew, Tsai,
D1135E/R1335Q/T1337R)
Topkar, et al. 2015)
Staphylococcus aureus Cas9 NNGRRT or NNGRR(N) (Plessis et
al. 1992)
Cas9 (“KKH”; NNNRRT (Kleinstiver, Prew, Tsai,
E782K/N968K/R1015H)
Nguyen, et al. 2015)
Neisseria meningitides Cas9 NNNNGATT (Hou et al. 2013)
Streptococcus Cas9 NNAGAAW (Sapranauskas et al.
thermophiles
2011)
Francisella novicida Cpf1 TTN (Zetsche et al. 2015)
Acidaminococcus sp. Cpf1 TTTN (Zetsche et al. 2015)
BV3L6
Lachnospiraceae Cpf1 TTTN (Zetsche et al. 2015)
bacterium
Alicyclobacillus C2c1 TTN (Shmakov et al. 2015)
acidoterrestris
IUPAC nucleotide code, where R refers to A or G; W to A or T; and N represents any nucleotide.
3. CRISPR-CAS9 GENE EDITING IN GENETIC

EYE DISEASE
The eye is an ideal site for testing of in vivo gene editing due to its
accessible position, compartmentalised anatomy and relative immune-
privilege. CRISPR-Cas9-based therapeutics provide the possibility for
permanent treatment of genetic disease. Inherited eye diseases affect
millions of people worldwide and have an immense socioeconomic impact.

With our increasing understanding of the underlying molecular
mechanisms of ocular diseases, gene therapy, especially combined with
adeno-associated viruses (AAV) delivery, has been proposed as an
effective approach. With the advantages of CRISPR/Cas over alternate
genome editing technologies, the stage is set for a new wave of gene-based
clinical trials. The benefits of using CRISPR/Cas9 systems to study eye
diseases have been highlighted by several successful applications in a
variety of cell types and animal species.
3.1. Applications for Retinal Eye Disease
Research into CRISPR/CAS9 gene editing as applied to ocular

diseases was initially focused on diseases that affect the retina. Hereditary
retinal diseases comprise the leading cause of blindness among working
age adults in the United Kingdom (Liew, Michaelides, and Bunce 2014).
Retinal diseases like proliferative diabetic retinopathy (PDR), retinopathy
of prematurity (ROP), and wet age-related macular degeneration (wAMD)
are known to occur from abnormal angiogenesis, the process by which new
blood vessels grow from pre-existing vessels (Abhinand et al. 2016).
Vascular endothelial growth factor (VEGFR) plays a critical role in
angiogenesis, and experiments utilizing CRISPR/Cas9 to edit VEGFR
have shown promise. Editing VEGFR2 in mouse models for oxygen-
induced retinopathy (OIR) and laser-induced choroid neovascularization
(CNV) was shown to abrogate angiogenesis (Huang et al. 2017). In
addition, another study showed the knockout of Vegfa and Hif1a genes by
CRISPR-Cas9 to block angiogenesis in a CNV model in mouse (Kim,
Park, et al. 2017). Subretinal injection of sgRNA- ribonucleoproteins
(RNPs) complexes into the adult mouse eye gave rise to mutagenesis at the
target Vegfa gene in the retinal pigment epithelium resulting in reduced
CNV in an AMD mouse model (Kim, Park, et al. 2017).
Targeted genomic deletion using the CRISPR/Cas9 system presents a
promising therapeutic approach for the treatment of patients with Leber's
congenital amaurosis 10 (LCA10) bearing the CEP290 splice mutation

(Ruan et al. 2017b). Loss of cone photoreceptors in RP leads to blindness,
and preservation of cone function is a major therapeutic goal. Sub-retinal
injection of AAV-delivered CRISPR/Cas9 prevented retinal degeneration
in mice by Nrl knockdown in rod cells (Yu et al. 2017). Furthermore, gene
editing using CRISPR/Cas9 has been shown to correct retinal dystrophy in
a rat model of autosomal dominant RP by generating allele-specific
disruption of RhoS334 (Bakondi et al. 2016).
3.2. Corneal Autosomal Dominant Genetic Eye

Disease–Allele Specificity
The majority of corneal dystrophies are the result of an autosomal

dominant inheritance pattern (Klintworth 2009). Others exhibit autosomal
recessive inheritance, while a few corneal dystrophies present X-linked
inheritance. To date, 23 distinct corneal dystrophies have been described.
Table 4 describes each corneal dystrophy, including; associated inheritance
pattern, gene locus and causative genes. Corneal dystrophies are
predominantly monogenic and highly penetrant. The accessibility of the
cornea, make the corneal dystrophies an ideal starting point for genome
editing therapies where concepts of personalized medicine can be put to
use (Moore et al. 2018).
Research into the use of CRISPR/Cas9 for the treatment of rare
inherited corneal disease has progressed during the past few years. We
were the first to demonstrate an allele specific CRISPR/Cas9 approach,
cleaving the mutant DNA at a SNP-derived PAM in the KRT12 gene in the
mouse cornea (Courtney et al. 2016). For this approach, we took advantage
of the discriminating nature of the PAM, the recognition site for the CAS
enzyme, which requires a precise sequence composition for the DSB to
occur. When a DNA mutation or single nucleotide polymorphism (SNP) is
known to cause disease also creates a PAM where none existed before, this
mutation can be exploited to precisely target the mutant allele with the
CRISPR/Cas system. In this study, a disease causing missense mutation,
p.Leu132Pro which generates a novel PAM site was identified for

Meesmann's epithelial corneal dystrophy (MECD) within the KRT12 gene
(Courtney et al. 2016). MECD most commonly occurs as an autosomal
dominant genetic disease, and we were able to knock down the mutant
allele via NHEJ while leaving the wild type allele intact. This approach of
allele specific gene editing could be used to treat other ocular dystrophies
caused by dominant negative missense mutations that generate novel
PAMs, further expanding the potential for CRISPR/Cas9-based
therapeutics.
Table 4. Corneal dystrophies: associated inheritance pattern, gene

locus and causative genes
Corneal layer Disease Inheritance Genetic Gene Gene/s IC3D Category

Pattern Locus known Affected
Epithelial and EBMD Minority of 5q13 Some cases TGFBI Some C1
Sub-Epithelial cases, mostly
Dystrophies sporadic
ERED Autosomal Unknown Unknown N/A C4
Dominant (Smolandiensis
variant = C3)
SMCD Autosomal Unknown Unknown Unknown C4
Dominant
MECD Autosomal 12q13 and Yes KRT3 and C1
Dominant 17q12 KRT12
(Stocker–
Holt variant)
LECD X- Xp 22.3 Unknown Unknown C2
chromosoma
l dominant
GDCD Autosomal 1p32. Yes TACSTD2, C1
Recessive previously
M1S1
Bowman RBCD Autosomal 5q13 Yes TGFBI C1
Layer Dominant
Dystrophies TBCD Autosomal 5q13 Yes TGFBI C1
Dominant
10q24 Unknown Unknown C2
GWCD Autosomal Unknown Unknown Unknown C4
Dominant
Stromal LCD Autosomal 5q13 Yes TGFBI C1
Dystrophies Dominant
Autosomal 9q34 Yes GSN C1
Dominant
GCD Autosomal 5q31 Yes TGFBI C1
Dominant
MCD Autosomal 16q22 Yes CHST6 C1
Recessive
Table 4. (Continued)
Corneal layer Disease Inheritance Genetic Gene Gene/s IC3D Category

Pattern Locus known Affected
SCD Autosomal 1p36 Yes UBIAD1 C1
Dominant
CSCD Autosomal 12q21.33 Yes DCN C1
Dominant
FCD Autosomal 2q35 Yes PIP5K3 C1
Dominant
PACD Autosomal 12q21.33 Yes KERA, C3
Dominant LUM,DCN,E
PYC
CCDF Unknown Unknown Unknown Unknown C4
PDCD Reported AD Unknown Unknown Unknown C4
Descemet's FECD Unknown 13pTel – Unknown Unknown C3 or C4
Membrane 13q12.13,
and 15q,
Endotehlial 18q21.2 –
Dystrophies q21.32.
Autosomal 1p34.3 – Yes COL8A2 C1
Dominant p32
PPCD Autosomal 20p11.2– Unknown Unknown C2
Dominant q11.2
1p34.3– Yes COL8A2 C1
p32.3
10p11.2 Yes ZEB1 C1
CHED Autosomal 20p 11.2– Unknown Unknown C2
1 Dominant q11.2
(pericentro
meric
region)
CHED Autosomal 20p13 Yes SLC4A11 C1
2 recessive (telomeric
portion)
XECD X- Xq25. Unknown Unknown C2
chromosoma
l dominant
Total: 23 Known = 20 Known = Known =
17 10
Partially
known = 4
Unknown =
3.3. Mutant Allele Targeting, a Dual Cut

Approach
There are at least 70 known missense mutations located in the coding

region of the TGFBI gene that can lead to various forms of corneal
dystrophy. It is neither possible nor realistic to design a sgRNA for each of
these mutations, and since at least 44 of the 70 mutations occur adjacent to
a naturally occurring PAM, stringent fidelity is required to achieve
complete allele-specificity to avoid the off target site that exists in the form
of the wild-type allele for these mutations (Christie et al. 2017). There have
been studies which demonstrated that truncating the sgRNA leads to an
increased target specificity and reduced off target activity (Pattanayak et al.
2013, Fu, Reyon, and Joung 2014, Fu et al. 2014). However, it has been
shown by our group that a range of truncated guide lengths or addition of
5′-GG to the 20 nt guide sequence did not provide a marked
improvement of specificity for a sub group of the TGFBI mutations
(Figure 2) (Christie et al. 2017). We therefore have conceived of a dual
cut approach that in theory would achieve knock down for all the TGFBI
missense mutations applicable to heterozygous genotypes (Christie KA
2019). With a dual cut approach, the treatment of autosomal disease like
TGFBI corneal dystrophy is not constrained by the specific mutation
responsible for any given phenotype. Our approach utilises natural
occurring variants in the target region characterized by SNP-derived
PAMs, which allow for the design of allele-specific SNP-derived PAM
sgRNAs (Christie KA 2019). Utilising natural variants in the target region
that harbour a PAM which also lie in cis with the causative mutation
allows this method to discriminate between the wild-type and mutant
alleles. Premature termination of translation via NHEJ brings on permanent
disruption of the target allele. A dual guide approach to CRISPR/Cas9
editing was shown to be a promising therapeutic approach for the treatment
of patients with LCA10 bearing the CEP290 splice mutation, (Ruan et al.
2017a) and in a human cell line it was demonstrated that DNA deletions up
to 10 kb coupled to precise NHEJ effectively knocked down a mutant

allele (Zheng et al. 2014). In order to generate a therapeutic outcome for
the TGFBI mutation targeted in our work, an in cis dual guide approach
was required (Christie KA 2019).
Figure 2. Guide-length screen to determine the effect on specificity of a guide-specific

system. Reports have indicated that truncating the length of the matching sequence
within the guide from 20 to 18 nucleotides can reduce genome-wide off-target cutting,
while maintaining on-target efficiencies. An assessment of the effect of guide-length
upon specificity between the wild-type and mutant alleles, using a dual-luciferase
assay, was conducted for the 5 most prevalent TGFBI mutations. Reports have shown
that guide lengths <16nt abolish cleavage activity. For each mutation a range of guide
lengths from 16 to 22 nucleotides were tested, each guide was targeted to the wild-type
and respective mutant sequence and the firefly luciferase activity was measured as an
indicator of specificity. For all mutations investigated, the truncated guides did not
provide a marked improvement of specificity; for most cases maximal discrimination
occurred with guides 20 or 19 nucleotides in length. For R124C, a 20nt guide seemed
to confer allele-specificity, however, no other guide length offered any adequate
discrimination (Figure 2a). In the case of the R555Q mutation, guides in the 18–20nt
range did not offer sufficient discrimination, although, interestingly, the 21nt guide
provided convincing allele-specificity (Figure 2d). R555W did not offer any
considerable allele-specificity for any length tested (Figure 2e). R124H and R124L
displayed clear allele-specific cleavage, especially in the 18–20nt sgRNA range, with
minimal cutting of the wild-type sequence (Figure 2b and c). Interestingly for the R124
mutations guide lengths of 21nt seemed to impair cleavage activity in all cases.
While our study focussed on the issue of on-target allele-specificity in

relation to the TGFBI corneal dystrophies, for translation to the clinic a
number of key hurdles will need to be overcome, including genome wide
specificity and efficiency of delivery to the correct cells in the cornea.
Potent targeting of the correct cell population must be achieved. In corneal
tissue, the majority of TGFBI protein is produced in the epithelium, and
the epithelium is continually turned over and repopulated via the limbal
epithelial stem cells (LESCs). Thus, in order to permanently correct the
TGFBI corneal dystrophies efficient delivery to the LESCs must be
achieved (Christie KA 2019).
Another application for utilizing a dual cut CRISPR/Cas9 approach in
the context of the cornea involved a study aimed at understanding the
function of the PAX6 gene in maintaining the identity of human corneal
epithelial cells (CECs) (Kitazawa et al. 2017). PAX6 is a transcription
factor and is necessary for eye development. To investigate how PAX6
regulates the gene expression profile of CECs, this study designed a two
guided RNA system targeting specific genomic loci to make a DSB that
resulted in a nonsense mutation upon NHEJ. This resulted in a complete
knock down PAX6 expression. The PAX6 knockout via the dual cut
CRISPR/Cas9 system resulted in a loss of CEC identity through the

downregulation of corneal epithelial-related genes and the upregulation of
epidermis and keratinization-related genes. Specifically, PAX6-depleted
CECs resulted in down-regulated genes that were primarily CEC-specific
and included keratin 12, keratin 3, clusterin (CLU), aldehyde
dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7
(ANGPTL7) and transketolase (TKT), while up-regulated genes were
primarily epidermis-related and included keratin 10, keratin 1, involucrin
(IVL) and filaggrin (FLG). The findings suggested that PAX6 maintains
CEC identity by regulating differentiation (Kitazawa et al. 2017).
3.4. Multiplexing in the CRISPR System for

Multigene Ocular Disease
Complex genetic ocular diseases like primary open angle glaucoma

(POAG) or AMD where dysregulation at multiple loci contribute to the
overall disease phenotype could be treated by multiplexing the CRISPR
system. Likewise, retinal dystrophies are genetically heterogeneous
diseases (Berger, Kloeckener-Gruissem, and Neidhardt 2010, Roosing et
al. 2014, Zeitz, Robson, and Audo 2015). An advantage of the
CRISPR/Cas system over protein recognition-based methods is its ability
to perform multiplex genome editing at high efficiencies by expressing or
providing multiple guide RNAs (Cong et al. 2013, Mali, Yang, et al. 2013).
Table 5 describes current approaches for providing multiple gRNAs in
vivo. More recently, multiplexing was achieved with AAV-delivered Cas9
nuclease. A quadruplex gRNAs/SaCas9 vector consisting of SaCas9 and
multiplex sgRNAs was successfully delivered using AAV-DJ/8 for in vivo
excision of HIV-1 proviral DNA in various solid tissues/organs via a single
intravenous injection in humanized bone marrow/liver/thymus (BLT) mice
with chronic HIV-1 infection (Yin, Zhang, et al. 2017).
A construct referred to as CRISPR-on was used in a study where an

exogenous reporter gene in mouse and human cells was activated enabling
the simultaneous activation of multiple endogenous genes (Cheng et al.
2013). The system utilized a nuclease-dead Cas9 (dCas9) protein fused
with a transcriptional activation domain and a single guide RNAs
(sgRNAs) with complementary sequence to gene promoters. The ability a
system like CRIRPR-on to regulate endogenous genes is valuable for the
study of gene functions and has great potential for therapeutic applications.
Another study found that by introducing different Cas9 proteins they could
mediate DNA cleavage or regulate transcriptional activity simultaneously
(Esvelt et al. 2013). Catalytically active Cas9 and distinct sgRNA
constructed with 14-15-bp target sequences and MS2 binding loops
activated gene expression without inducing DSBs. Co-expression of full
length normal sgRNA and truncated sgRNA fused with MS2 binding loops
can simultaneously cleave or activate transcription at the respective target
genes (Dahlman et al. 2015).
Table 5. Current approaches for providing multiple gRNAs
Multiplex CRISPR System Description

Multigene cassettes to express A lentiviral system was used to express a Cas9 variant, a
several gRNAs reporter gene and up to four sgRNAs from independent RNA
polymerase III promoters incorporated in the vector with the
Golden Gate cloning method. Each sgRNA was efficiently
expressed and was able to mediate multiplex gene editing
(Kabadi et al. 2014).
The expression cassettes of gRNAs were tandemly ligated into a
single vector using the Golden Gate cloning method.
Additionally, Cas9 nickase-mediated genome editing was
constructed with a vector expressing Cas9 nickase and six
gRNAs targeting three genomic loci (Sakuma, Sakamoto, and
Yamamoto 2017).
Guide RNAs post- An RNA-based regulation and CRISPR/Cas transcription factors
transcriptionally processed (CRISPR-TFs) tool utilizing multiple RNA regulatory strategies,
from a single transcript using including RNA-triple-helix structures, introns, microRNAs, and
Csy4-based excision ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based
RNA processing gives the ability to modulate endogenous
promoters including multistage cascades and RNA-dependent
networks (Nissim et al. 2014).

tRNA-dependent cleavage of A general strategy to produce numerous gRNAs from a single
gRNAs polycistronic gene was utilized to boost the targeting and
multiplex editing capability of a CRISPR/Cas9 system. The
study demonstrated that synthetic genes with a tandemly arrayed
tRNA–gRNA architecture, efficiently and precisely processed
gRNAs to desired 5′ targeting sequences in vivo and directed
Cas9 to edit multiple chromosomal targets (Xie, Minkenberg,
and Yang 2015).
Arrays to express multiple Using an artificial CRISPR pre-crRNA array consisting of four
crRNAs for Cpf1 spacers separated by direct repeats (DRs) from the CRISPR
locus of Francisella novicida (FnCpf1) and two Cpf1 orthologs
with activity in mammalian cells, Acidaminococcus Cpf1
(AsCpf1) and Lachnospiraceae Cpf1 (LbCpf1) multiplex gene
editing was observed (Zetsche et al. 2017).
Self-cleaving ribozyme Multiple gRNAs linked with self-cleaving ribozymes and/or
flanked gRNAs tRNA could be simultaneously expressed from a single U6
promoter to exert genome editing. The study provided a novel
strategy to enable tissue-specific expression of more than one
gRNAs for multiplex gene editing (Xu et al. 2017).
Multiplex CRISPR System Description

Direct introduction of Cas9 A method for the rapid synthesis of gRNA and for delivery of
proteins preloaded with Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs)
different gRNAs Cas9 RNPs into a variety of mammalian cells through liposome-mediated
transfection or electroporation was described. This approach for
multigene targeting in Jurkat cells resulted in two-locus and
three-locus indels in approximately 93% and 65% of the
resulting isolated cell lines with a reduced off-target cleavage
rate (Liang et al. 2015).
4. DELIVERY OF CRISPR/CAS TO THE EYE -

IN VIVO AND EX VIVO
Figure 3. Delivery of Cas9-sgRNA complex. A. Viral vector based delivery B.

Physical method of microinjection based delivery C. Nanoparticle based delivery.
Efficient and effective delivery of the CRISPR/Cas machinery to the

tissues or cells of interest is one of the most important aspects for
translating the therapy to the clinic. The physiology of the eye presents
challenges for the pharmacologist and formulation scientist for the design
of efficient drug delivery systems. Delivery of drugs to the targeted ocular
tissues is restricted by various precorneal, dynamic and static ocular
barriers (Patel et al. 2013). The outermost surface of the eye has many
barriers that prevent entry of a foreign substance. Biological barriers must
be considered for any CRISPR/Cas system to be delivered to the eye.
Many avenues of delivery have been explored, broadly sub-divided into
viral and non-viral approaches (Figure 3). An ideal vehicle for gene
therapy should have a large capacity to package all the CRISPR/Cas9
machinery, cell tropism for the specific cell population of interest, high
nucleus targeting efficiency along with minimal immune response and
cytotoxicity.
4.1. In Vivo Systems

A range of viral systems including adenoviruses, AAVs and

retroviruses, specifically lentivirus, has been investigated for their potential
to deliver genetic payloads to transduce various types of tissues, including
the eye (Kay 2011). Adenovirus has the advantage of a large packaging
capacity but initiates a substantial immune response; (Borras et al. 2001,
Tsubota et al. 1998) whereas, AAV provides efficient transduction with a
minimal immune response and persistence in the nucleus but has a limiting
packaging capability (Sharma et al. 2010, Zinn and Vandenberghe 2014).
Lentivirus has a considerable packaging capacity but can cause unwanted
integration (Athanasopoulos, Munye, and Yanez-Munoz 2017). Non-
integrating viruses like adenoviruses transiently express the transgene
making them ideal for applications with CRISPR/Cas system, as limited
Cas9 expression reduces the off target cleavages.
AAV has been the leading vector of choice for gene therapy,
particularly ocular gene therapy. Recently AAV technology has been
deployed for delivering CRISPR/Cas9 components to investigate gene
function and disease treatment in the mammalian eye (Huang et al. 2017,
Ruan et al. 2017b, Yu et al. 2017, Kim, Koo, et al. 2017). AAV has been
shown to transfer genes successfully into photoreceptor cells and retinal
pigment epithelium (RPE) of the mouse retina with low cytotoxicity (Day
et al. 2014). Sub-retinal injection of an AAV vector has been shown to be
safe and effective in animal studies, (Acland et al. 2001, Pang et al. 2006)
and human trials (Boye et al. 2013, Cideciyan et al. 2009, Jacobson et al.
2012, MacLaren et al. 2014, Testa et al. 2013, Trapani et al. 2015, Pierce
and Bennett 2015). The use of AAV-2 based gene augmentation therapy
for RPE65-associated retinal degeneration gene therapeutics has been
extensively tested in clinical trials (Pierce and Bennett 2015). However,
AAV-2 was found to be not as effective as other AAV serotypes for
transduction within the cornea, and as such a variety of other AAV vectors
have been shown to successfully transduce the cornea with varying success
(Sharma et al. 2010, Liu et al. 2008). A study reported that a hybrid
serotype, AAV-2/8, delivered by an intrastromal injection was able to
transduce long term expression of the transgene in the stromal keratocytes
in both ex vivo human and in vivo mouse models (Hippert et al. 2012). An
intracameral injection of the AAV hybrid serotype vector, AAV-2/9 was

shown to transduce corneal endothelial cells in mouse (O'Callaghan et al.
2017). In our hands AAV- 2/9 was effective in transducing both the
corneal epithelium and corneal endothelium cells (Unpublished Data).
Lastly, a novel synthetic AAV has been reported to efficiently transduce
corneal stroma and endothelial cells following intrastromal injection in
mouse (Wang et al. 2017).
With AAV vectors being the leading choice of gene therapy in
ophthalmology, different strategies have been applied to circumvent their
limiting packaging capacity. A dual AAV vector system was utilized to
deliver the the CRISPR components, the sgRNA and SpCas9 in two
separate AAV vectors and delivered in vivo to post mitotic photoreceptor
cells in experiments utilizing retinal degeneration mouse models (Yu et al.
2017). Since the crystal structure of at least two Cas9 proteins complexed
with guide RNA and target DNA have been determined, (Nishimasu et al.
2014, Yamano et al. 2016) the way has been paved for designing
modifications to the complex which could be used to overcome the
payload restrictions of the AAV vectors. Also, to limit an AAV mediated
gene therapy’s persistent transgene expression, the use of tissue-specific
promoters or self-cleaving vectors have been investigated. A tissue-
specific reporter ensures expression is localised to only the desired target
tissue. In 2 clinical trials where LCA was treated with gene replacement of
RPE65, an AAV-2 vector with a human RPE65 promoter, hRPE65 was
used to minimise unwanted expression in other tissues (Bainbridge et al.
2008). With respect to gene editing with a CRISPR system, minimising
expression of Cas9 may help to reduce off-target effects (Moore et al.
2018). The use of a self-cleaving system which involves introducing both a
sgRNA targeted to the gene of interest and a sgRNA targeted to the
delivery vector could also be used to minimise the period of transgene
expression and off-target effects (Chen et al. 2017, Moore et al. 2014,
Petris et al. 2017).
As an alternative to non-viral delivery systems, broadly classified into
two sub categories are physical and chemical systems (Ramamoorth and
Narvekar 2015). Unlike viral vectors, non-viral systems are not limited by
packaging restrictions, and they have lower immunogenicity and toxicity

(Pack et al. 2005, Mintzer and Simanek 2008). Physical methods including
injection of naked DNA, electroporation, gene gun, ultrasound and
magnetofection utilise force to increase cell permeability (Mohan et al.
2005, Oliveira, Rosa da Costa, and Silva 2017). Chemical delivery
methods employ the principal of electrostatic interactions between the
negatively charged nucleic acid and either a natural or induced positive
charge of the carrier, generating a complex with an overall positive charge
(Jin et al. 2014). Through carrier design alteration and by targeting the
ligand decoration on the cell types, non-viral vectors can deliver
therapeutic nucleic acids to target tissues or cells. Several naturally
occurring and synthetic cationic polymers have been used for the delivery
of nucleic acids. A novel hyaluronic acid-chitosan nanoparticle was used to
transfect efficiently the corneal and conjunctival cells in rabbits in vivo and
in a human in vitro model, (de la Fuente, Seijo, and Alonso 2008a, b) and
ultrapure chitosan oligomers were used as carriers for transferring a
luciferase plasmid to keratocytes in rat cornea (Klausner et al. 2010).
Interestingly, another report developed an artificial virus using a
nanoparticle, liposome-protamine-DNA complex (LPD), modified with a
cell permeable peptide and a nuclear localization signaling (NLS) peptide,
to deliver a functional Rpe65 gene to mouse eyes improving vision in vivo
(Rajala et al. 2014). Thus, LPD nanoparticles provide a promising,
efficient, non-viral method of gene delivery with clinical applications.
Other promising delivery systems utilising the advantages of non-viral
delivery are CRISPR/Cas9 RNP complexes (Zuris et al. 2015) and
CRISPR-Gold which is able to simultaneously deliver RNPs and donor
DNA into cells resulting in successful in vivo HDR (Lee et al. 2017). A
recent work demonstrated that a structure-guided chemical modification of
guide RNA delivered using a non-viral lipid nanoparticle resulted in a
>80% gene editing of Pcsk9 (Yin, Song, et al. 2017). The advantage of
using a direct protein-packaged approach is that components of the
CRISPR complex have a short half-life and are not being continuously
synthesized in the cell once the genomic modification has occurred. As no
delivery system on its own meets all requirements, therapeutic genome
editing could be best achieved by combining a viral coupled to non-viral

delivery. For example, Cas9 mRNA delivered using lipid nanoparticles
along with AAV encoding a sgRNA and a repair template has been shown
to induce repair of Fah in a mouse model (Yin et al. 2016). These studies
pave the way for more efficient use of alternative in vivo delivery systems
for gene editing especially in the eye.
4.2. Ex Vivo Systems
Stem cell therapies for the treatment of AMD and other degenerative
retinal disorders including inherited retinal disease are positioned to make
an impact on clinical care (Wood et al. 2019). Stem-cell-based
photoreceptor cell replacement has been investigated in different cell types
and in animal models. The CRISPR/Cas9 system was successfully
employed in embryonic stem cells (ESCs) where efficient gene correction
was demonstrated in a mouse cataract model (Wu et al. 2013). A study
utilizing human embryonic stem cells (hESCs) demonstrated the ability of
a hESC-derived retina to form structured mature photoreceptor layers after
transplantation into nude rats and two different monkey models (Shirai et
al. 2016). Human retinal progenitor cells (hRPCs) from fetal neural retina
were transplanted into a rat model where at 12 weeks after treatment,
hRPC-grafted eyes had significantly superior visual acuity compared to
non-treated eyes (Luo et al. 2014). For the study of optic nerve biology and
disease, a CRISPR engineered reporter cell line was able to demonstrate
the differentiation of hESCs to retinal ganglion cells (RGCs) (Sluch et al.
2015). Although hESCs offer extensive differentiation capabilities
allowing for the study of very early stage development of life as well as
providing a source of cells for the development of novel therapeutic
molecules and cell-based therapies, their use is controversial. Due to the
ethical and practical reasons of using cells derived from embryonic or fetal
tissue, much work and effort has been conducted with patient-derived
induced pluripotent stem cells (iPSCs), which offer the best possible
immunologic match to the patient (Burnight et al. 2017).
The use of iPSCs in gene editing technologies hold great promise in

the treatment of inherited disease. CRISPR/Cas tools enable precise
manipulation of these stem cells for ophthalmic applications providing a
platform for correction of deleterious mutations in a patient's own cells.
For three different types of variants that lead to retinal diseases,
CRISPR/Cas9 mediated genome editing strategies were used to target and
correct the mutations in patient-derived iPSCs. A HDR strategy targeting
an Alu insertion in the MAK gene was able to restore the retinal transcript
and protein; the CRISPR-Cas9-mediated NHEJ approach was able to
excise the IVS26 cryptic-splice mutation in CEP290 demonstrating a
correction of the transcript and protein in patient iPSCs; and lastly, with
allele-specific CRISPR guides, the Pro23His rhodopsin (RHO) allele was
selectively targeted which, following delivery to both patient iPSCs in
vitro and pig retina in vivo, created a frameshift and premature stop
preventing transcription of the disease-causing variant (Burnight et al.
2017). Additionally, in patient-specific iPSCs an RPGR point mutation that
causes X-linked RP was precisely repaired (Bassuk et al. 2016).
While iPSC driven ex vivo therapy for inherited corneal disease has not
yet been realized, the potential and the development of cell-based therapies
using stem cells represent a significant breakthrough in treatment options
in regenerative medicine for the ocular surface. There have been several
studies focused on differentiating iPSCs into corneal epithelial cells for the
treatment of limbal stem cell deficiency (LSCD) (Saghizadeh et al. 2017).
One such study was able to successfully induce corneal epithelial cells
from both human adult corneal limbal epithelial cells (HLEC)-derived iPS
cells and human adult dermal fibroblast (HDF)-derived iPS cells by the
stromal cell-derived inducing activity (SDIA) differentiation method
(Hayashi et al. 2012). Another study was able to derive iPSCs from human
primary LSCs and re-differentiate these iPSCs back into the limbal corneal
epithelium (Sareen et al. 2014). Our group previously reported the ability
to culture LESCs from a patient with MECD. The causative mutation,
p.Leu132Pro in the KRT12 gene in these cultures was effectively silenced
using siRNA (Courtney, Atkinson, Allen, Moore, Walsh, Pedrioli,
MacEwen, Pellegrini, Maurizi, and Serafini 2014).
On-going research with corneal cell models has advanced the potential
for the CRISPR/Cas9 complex to be delivered ex vivo for the treatment of
inherited corneal disease like the TGFBI corneal dystrophies. Efficient
corneal transduction resulting in transgene expression via intra-stromal
injection of an AAV2/8 vector was successful in human corneal explants
(Hippert et al. 2012) An AAV8/9 chimeric capsid was also shown to
transduce human corneal explants in a gene replacement study for the
treatment of cornea clouding brought on by mucopolysaccharidosis type 1
(MPS1) (Vance et al. 2016). While an AAV approach to cellular
transduction has shown promise, there are alternative methods to AAV and
viral transduction. The variant within the TGFBI gene that causes granular
corneal dystrophy type 2 (GCD2) was repaired in human corneal
keratocytes via a CRISPR/Cas9-induced HDR mechanism (Taketani et al.
2017). In this work a plasmid vector, px458 which enabled bicistronic
expression of SpCas9 and green fluorescence protein (GFP) was co-
transfected into primary GCD2 mutant human corneal keratocytes with a
single stranded 100 base oligonucleotide (ODN) that served as donor repair
template. This study was the first to demonstrate in vitro gene correction in
mutant human primary corneal cells using CRISPR/Cas9 and HDR
(Taketani et al. 2017).
The importance of an ex vivo approach to the delivery of therapeutic
cells containing the CRISPR/Cas9 complex (Figure 4) for the treatment of
autosomal dominant disease such as GCD2 and other TGFBI corneal
dystrophies is crucial when NHEJ resulting in a knock down of the mutant
allele is not a viable option. We have established that the TGFBI protein is
necessary for the wound healing process (Christie KA 2019). As such,
individuals who carry homozygous mutations for an autosomal dominant
TGFBI corneal dystrophy cannot be treated with allele specific
CRISPR/Cas9 NHEJ gene editing since, with this method both alleles
expressing the TGFBI transcript would be silenced. HDR within a corneal
stem cell model could potentially be used to replace mutant corneal cells
within an individual who carries the mutation in both copies of the TGFBI
gene. However, ex vivo stem cell transplantation of gene edited corrected
cells via HDR is dependent on removing all resident mutations carrying
LESCs, and for those TGFBI mutations resulting in an intrastromal

pathology, stem cell transplantation will not undo existing stromal
pathology. As such, a corneal dystrophy such as MECD which affects the
anterior corneal epithelium (McLean and Moore 2011) is well suited for ex
vivo stem cell replacement. This approach would be most advantageous for
targeting the MECD SNP-derived PAM mutation, p.Leu132Pro in the
KRT12 gene (Courtney et al. 2016).
Figure 4. CRISPR therapeutic approach; transition to clinics.
5. ANIMAL MODELS FOR EYE DISEASE
The generation of new model systems with genome-editing tools, in

particular those based on CRISPR/Cas9, are opening new avenues for both
research into, and for, the treatment of eye disease. Genome-editing
technologies are allowing researchers to take full advantage of both animal
and cellular models, and to work more easily with non-traditional model
organisms for eye research. In ophthalmology, these animal models are
important first steps for examining genetic conditions as well as assessing
therapeutics for such genetic diseases. For generating a transgenic animal
model, the CRISPR/Cas9 system has multiple advantages over the ZFN or
TALEN approach to gene editing. The gRNA determines the specificity of
CRISPR systems and not the nuclease; therefore, the CAS protein can
remain unchanged between target sites and between different organisms.
Adjusting the target with a change to the gRNA sequence allows for higher
efficiencies when compared to systems that require protein alterations or
changes to embryonic stem cells (ES). Since homologous recombination is
generally required to insure a site‐specific edit within ES cells, and given
that a DSB can increase the probability of homologous recombination,
CRISPR/Cas9 increases specificity and site‐specific genome targeting
frequency in view of the system’s effectiveness in producing DSBs (Zarei
et al. 2019). The emergence of this technology has allowed the genome
editing in mouse embryos to be quicker and more cost-efficient (Yang et
al. 2013). In addition, CRISPR/Cas9 has revolutionized the generation of a
wide variety of transgenic animals such as zebrafish, (Ablain et al. 2015)
rats, (Bakondi et al. 2016) monkeys, (Zuo et al. 2017) pigs, (Yan et al.
2018) and rabbits (Yuan et al. 2016).
Zebrafish represent a powerful model for studying vertebrate eye
development. Easy access to eye tissue for imaging and drug/gene
delivery, short lifecycle, and genetic similarities to higher vertebrates and
mammals gives zebrafish many advantages over other model organisms to
explore the applications of CRISPR/Cas gene editing in the study of
important pathways and regulators in the development of ocular tissue
(Richardson et al. 2017). Several studies demonstrate the power of
CRISPR/Cas genome editing in zebrafish. CRISPR/Cas gene editing was
applied in the zebra fish model to understand the role of membrane frizzled
related protein (MFRP) in the development of nanopthalmic-hyperopia;
(Collery et al. 2016) SpCas9-generated knockouts and morpholinos to
show the importance of NeuroD in photoceptor regeneration; (Taylor et al.
2015) and to show retina regeneration and Müller glia de-differentiation
(Yin et al. 2015, Serifi et al. 2016). In another non-mammalian animal
model, an amphibian, Xenopus tropicalis was used to create a highly
penetrant and rapid retinoblastoma (Rb) model. CRISPR/Cas9 gRNAs
were injected into X. tropicalis embryos for rapid identification of targets
for therapeutic intervention (Naert et al. 2016). These studies demonstrate
the important role of CRISPR/Cas genome editing in animal models like
the zebrafish for investigating diseases of the human eye.
CRISPR/Cas systems are also applied to mammalian models of the

eye. The following examples focus on research conducted with mouse
models for the treatment of different eye diseases. In a study of a particular
LCA model, CRISPR-engineered mosaicism reveals that the loss of
Kcnj13 function in mice mimics the human disease phenotypes (Zhong et
al. 2015). RP is a challenging retinal dystrophy for ophthalmologists. A
mouse model mimicking clinical phenotypes of RP was generated using
CRISPR like knock-in with the p.Leu135Pro, an RP-associated variant
(Arno et al. 2016). The most commonly studied preclinical model of RP
called the “rodless” (rd1) mouse is homozygous for two mutations, a
nonsense point mutation (Y347X) and an intronic insertion of a leukemia
virus (Xmv-28) (Wu et al. 2016). Utilizing a CRISPR/Cas9 system, it was
demonstrated that the Y347X mutation is the causative variant in this form
of RP. This group was the first to demonstrate homology-directed
recombination–mediated gene correction in the visual system using the
CRISPR/Cas9 system (Wu et al. 2016). Our research on MECD utilized a
humanized mouse containing a mutant KRT12 allele mutation,
p.Leu132Pro (Courtney et al. 2016). Through CRISPR/Cas9 directed
NHEJ repair resulting in frame-shifting deletions within the mutant KRT12
allele, we were able to demonstrate reduction of the mutant KRT12 mRNA
expression. The CRISPR/Cas9 system has also been successfully
employed in stem cell lines where efficient gene correction was
demonstrated in a mouse cataract model (Wu et al. 2013).
Pre-clinical studies in other CRISP/Cas9 edited mammalian models
include rabbits and pigs. A group studying GJA8, a gene known to be
involved in maintaining lens opacity and proper lens development, created
a gene knockout via a co-injection of Cas9/sgRNA mRNA into rabbit
zygotes. Their results showed that gene mutation efficiency in the GJA8
locus reached 98.7% in the rabbit embryos and that impaired GJA8
function caused microphthalmia, small lens size and cataracts; knocking
out GJA8 in rabbit via CRISPR/Cas9 system caused human-like cataracts
(Yuan et al. 2016). In research involving neurodegenerative Huntington’s
disease (HD), a large CAG repeat (150 CAGs) via somatic cell nuclear
transfer (SCNT) in combination with CRISPR/Cas9, was inserted into the
endogenous pig HTT gene generating a HD knockin (KI) pig model that
expressed full-length mutant HTT at the endogenous level (Yan et al.
2018). HD results from this monogenetic CAG repeat expansion located in
exon1 of the HTT gene. There are advantages to using large mammals such
as pigs over rodent and rabbit models. Pigs are genetically, anatomically,
and physiologically closer to humans, and their fast breeding period and
large litter size hold advantages over non-human primates when
considering the time line of generating large animal models of human
diseases (Yan et al. 2018).
6. RECENT PROGRESS IN CLINICAL GENE THERAPY

TRIALS FOR OCULAR DISEASES
Research for transitioning effective gene therapy strategies for ocular

disease to the clinic has rapidly grown in the past 15 years, particularly for
the treatment of retinal diseases. Clinical trials involving retinal gene
therapy are creating hope for future therapies for afflicted patients. They all
employ gene augmentation for loss of functions diseases, rather than gene
editing strategies. The most successful example of ocular gene therapy is
by gene replacement using AAV delivery (Pierce and Bennett 2015). It has
been used successfully for RPE65, Leber's congenital amaurosis 2 (LCA2),
an early onset form of autosomal recessive retinal degeneration caused by
mutations in the RPE65 (RPE-specific 65 kDa protein) gene. Ground
breaking trials utilising AAV vectors to deliver RPE65 did show safety and
initial vision improvements (Bennett et al. 2016, Jacobson et al. 2015).
These studies were the first to demonstrate proof of concept for gene
therapy in human retinal disease. Three separate phase I–II clinical trials
were initiated, which yielded promising results after subretinal
administration of AAV2-hRPE65 vectors (Bainbridge et al. 2008,
Hauswirth et al. 2008, Weleber et al. 2016). The three studies differed in
terms of dose, inclusion criteria, type of promoter, location of the injection
and outcome measures. All the studies reported high levels of safety and
demonstrated efficacy up until the 3-year follow-up. In 2018 subsequent
results were reported for the trial involving the AAV vector expressing
RPE65 (rAAV2-CB-hRPE65) (Weleber et al. 2016, Pennesi et al. 2018).
The outcome after 5 years was found to be small and not sustained;
however, there were no clinically significant adverse events. The group
concluded that treating patients at a younger age is associated with better
visual function outcomes during 5 years after treatment (Pennesi et al.
2018). In a follow up study for the trial administering the rAAV2/2 RPE65
vector, (Bainbridge et al. 2008) results indicated that the amount of
rAAV2/2 RPE65 required to drive the visual cycle in affected persons was
not met to the extent required for a durable, robust effect (Bainbridge et al.
2015). Gene therapy for LCA is the first ocular gene therapy to complete a
phase III clinical trial for patients with confirmed biallelic RPE65
mutation-associated retinal dystrophy (Jiang, Xu, and Tsang 2018).
Another gene therapy presently at the pre-clinical trial stage, targets
wAMD with an AAV vector encoding aflibercept (ADVM-022) (Grishanin
et al. 2019). Vascular endothelial growth factor (VEGFA) plays a key role
in the development of wAMD, and as such VEGFA is targeted in the
treatment of the disease. Administering recombinant anti-VEGFA proteins
such as aflibercept with intravitreal (IVT) injections every 4 to 8 weeks is
an approved therapy for this disease. Aflibercept is a recombinant chimeric
protein consisting of the VEGFA binding portion of human VEGFR-1 and
VEGFR-2 fused to the Fc portion of human IgG1 immunoglobulin (Ashraf
and Souka 2017). ADVM-022 utilizes a variant of the AAV2 capsid as a
vector for delivering and encoding aflibercpt. It was reported that the
administration of ADVM-022 in non-human primates was tolerated with
no serious adverse safety-related findings and that more than one year past
a single IVT injection, ADVM-022 continued to provide robust aflibercept
expression. The effects of the ADVM-022 treatment were found to be
similar to a bolus treatment of aflibercept protein (Grishanin et al. 2019). A
single dose injection has clear advantages over a frequent dosing schedule
where lack of patient compliance may result in recurrence of active
wAMD. Gene-based therapy for wAMD has the potential to overcome
such compliance issues by ideally providing a life-long supply of the anti-
VEGFA protein following a single vector administration.
The remarkable success of the RPE65 and ADVM-022 studies has

paved the way for other clinical trials, particularly using AAV-mediated
gene delivery; however, there are challenges in delivering an effective and
enduring dose to stop cell death or degeneration. Active clinical gene
replacement trials are underway targeting other retinal diseases such as
Stargardt's disease, RP, Usher syndrome, X-linked retinoschisis (XLRS),
choroideremia, achromatopsia, AMD and Leber hereditary optic
neuropathy (LHON) (Table 6). These trials use either AAV or lentivirus as
viral vectors.
Table 6. Current clinical trials of gene therapy in eye
Disease Phase of clinical Delivery Gene therapy Study plan and results
trial strategy
Leber's Multiple Phase I/II Sub retinal AAV2-hRPE65 Patients in all trials
congenital trials for RPE65- vector carrying exhibited several aspects of
amaurosis 2 associated LCA RPE65 gene visual improvements within
(LCA2) have been either a few months after
completed or are treatment, though with
ongoing. varying degrees
(Bainbridge et al. 2008,
Bennett et al. 2016,
Weleber et al. 2016,
Hauswirth et al. 2008).
trial strategy
Phase III clinical Sub retinal SPK-RPE65 An on-going study.

trial
(Jacobson et al. 2015)
Stargardt 48 week Phase Sub retinal Lentiviral vector No significant changes in
(Sanofi) I/IIA dose- injection SAR422459, visual acuity in either the
escalation trial carrying the treated or untreated fellow
photoreceptor eyes for the enrolled 23
specific ABCA4 patients. To continue
gene. enrollment in the cohort of
youngest patients with
early-onset Stargardt
disease and evidence of
rapid progression of disease
(ages 6 to 26 years; all
other cohorts involve

patients 18 years or older)
(Parker et al. 2016).
MERTK-RP Phase I study Sub retinal AAV2 vector Study recently completed in
with an RPE- Saudi Arabia. 6 patients
specific were treated without any
promoter serious adverse events.
driving MERTK Three of these patients
displayed measurably
improved visual acuity in
the treated eye following
surgery, although two of
them had lost that
improvement by two years
(Ghazi et al. 2016).
Usher1B 48 week Phase Sub retinal Lentiviral vector Out of the 9 adult patients
I/IIA dose- SAR421869 treated, a majority have
escalation trial (UshStat), shown an initial
carrying Myosin postoperative drop in
7A gene BCVA and visual fields
that improved to baseline
within two weeks. The
vision was stable (in either
the treated or untreated
fellow eyes) after 48 weeks
in a majority of patients.
(Unpublished Data).
trial strategy
X-linked 48 week Phase Intravitreal AAV8-RS1 A study evaluating three
Retinoschisis I/IIA safety and vector carrying different increasing dose
(XLRS) efficacy trial the gene levels of an AAV8-RS1
retinoschisin 1 vector in 9 participants with
(RS1) pathogenic RS1 mutations
with VA of 20/63 or worse
in one eye (NCT02317887)
(Cukras et al. 2018)
The second study is
evaluating an AAV-RS1
vector in up to 27 patients.
It involves three initial
groups of adult patients
receiving increasing dose
levels of the vector and will

also evaluate the maximum
tolerated dose level in
patients 6 years and older
(NCT02416622).
Choroideremia Multiple Phase I Sub retinal AAV-REP1 In all patients, the increase
and II trials of the vector carrying in retinal sensitivity over
AAV.REP1 vector RAB escort six months in the treated
are ongoing at protein 1 gene eyes correlated with the
several sites vector dose administered
per area of surviving retina.
(Cideciyan et al. 2009). The
early improvement
observed in two of the six
patients was sustained at
3.5 years after treatment
despite progressive
degeneration in the control
eyes. (Edwards et al. 2016)
Other trials of subretinal
placement of the
AAV.REP1 vector are
ongoing, including a Phase
I/II trial (NCT02341807).
trial strategy
Achromatopsi Phase I/II dose- Sub retinal AAV-CNGB3 The study is going on in
a escalation study vector carrying patients with CNGB3
CNGB3 gene achromatopsia at four sites
in the United States
(NCT02599922).
Age-related Phase 1 study Intravitreal AAV2-sFLT01 The treatment was safe
Macular with no dose-limiting
Degeneration toxicity (Heier et al. 2017).
(AMD)
Leber Two Phase 3 Intravitreal rAAV2/2-ND4 A study for patients with
Hereditary trials, in Europe (GS010) vision loss of six months or
Optic and the U.S less (NCT02652767), and
Neuropathy evaluating the another study is assessing
(LHON) effectiveness of patients whose vision loss
GS010 in early began more than six
onset LHON months but less than a year
patients ago (NCT02652780).
7. CRISPR/CAS GENE EDITING TRANSITION TO

CLINICS
CRISPR/Cas9 gene editing has undoubtedly shown great promise for

applications in gene correction therapy. The most imminent reason for
transitioning CRISPR/Cas research to clinics is direct therapeutic gene
editing in human cells, an advantage over viral-mediated gene therapy.
While no ocular gene therapy trials using CRISPR are yet underway, a
CRISPR gene editing technique has been approved by the National
Institutes of Health, US (NIH) to help extend cancer therapies that rely on
enlisting a patient’s T cells. Early in 2018 doctors at the University of
Pennsylvania were in the final steps of preparing for a clinical trial, and in
2019 two cancer patients who are enrolled in this study were treated
(Dearment 2019). This trial is small and designed to investigate whether
CRISPR is safe for use in people. In the study, doctors will remove
people’s blood cells, modify them with CRISPR in the lab, and then infuse
them back into the patients. The study involves three different types of
cancer: multiple myeloma, sarcoma, and melanoma and proposes to
remove T cells from at least 18 patients. Led by Dr. Edward Stadtmauer,
the protocol reprograms a person’s immune cells to find and attack tumors.
University of Pennsylvania scientists intend to use CRISPR to edit two
genes in patients’ T cells to make them better cancer fighters. One of the
genes to be edited, PD-1 makes a molecule that cancer cells utilize to block
the immune system from fighting the cancer. An additional edit removes a
natural T-cell protein that could interfere with this process and in its place,
an engineered receptor will steer the immune cells toward particular
tumors (Baylis and McLeod 2017) (Figure 4).
Similar studies have been under way in China where another trial is
making the first-ever attempt to use CRISPR to edit cells in the human
body infected with the human papillomavirus (HPV) while leaving the
DNA of normal cells untouched. (Figure 4). CRISPR would target and
destroy the viral genes of HPV that cause tumour growth. This study is to
begin at the First Affiliated Hospital of Sun Yat-Sen University in China
(Le Page 2017). In addition to this study, a research team in China edited
the embryos of twin girls born in November, 2018. In a highly
controversial undertaking, a team at the Southern University of Science
and Technology in Shenzhen purportedly eliminated the gene CCR5 via
CRISPR editing in the hopes of rendering the offspring resistant to HIV,
smallpox, and cholera. If proven successful, this effort would mark a
stunning medical achievement; however, this study and any future studies
of this kind are ethically charged because changes to an embryo could
eventually affect the entire gene pool if future generations were to inherit
the edited gene. It goes without saying, researchers and medical
practitioners should adhere to ethical guidelines such as those put forward
by the Academy of Sciences in the US. Currently, the use of genetically
engineered embryos to establish a pregnancy is prohibited in much of
Europe, the United States and China (Regalado 2018).
The use of CRISPR is also under development for treating ocular
diseases. Editas Medicine and Allergan Pharmaceuticals International
Limited (Allergan) plan to initiate patient screening for clinical trials to test
the efficacy of EDIT-101, a CRISPR genome editing drug for the treatment
of Leber congenital amaurosis 10 (LCA10). They plan to enroll 10-20
patients in the U.S. and Europe. Utilizing an AAV5 viral vector that
encodes for the Staphylococcus aureus Cas9 along with two different
gRNAs targeted to either side of the IVS26 mutation between exon 26 and
exon 27, EDIT-101 is hypothesised to delete or invert the mutation such
that functional CEP290 expression is restored. Recently published results
detail the development of EDIT-101 and summarizes in vitro experiments
in human cells and retinal explants (Maeder et al. 2019). Subretinal
delivery of EDIT-101 was well tolerated and achieved sustained CEP290
editing in photoreceptor cells in mice and non-human primates, which met
or exceeded the target therapeutic level. This study also determined that the
2 gRNAs chosen for this treatment were highly specific, and no off-target
sites could be verified in either multiple human cell lines or human retinal
cells. Furthermore, the results presented in this study were encouraging
since editing rates were above the therapeutic threshold for both mice and
non-human primates, thus providing the basis for dose extrapolation to
first-in-human trial for a CRISPR/Cas9 gene therapy. These clinical trials
would pave the way for a new era for treating the inherited ocular diseases
for which current disease prognosis is poor.
CONCLUSION
Gene editing holds enormous potential for the clinical treatment of

inherited eye diseases. The eye, being an ideal target for gene editing puts
ophthalmology at the forefront of the emerging CRISPR/Cas technology.
Improvements in the development of more efficient delivery methods and
more precise CRISPR/Cas components will allow these therapies to
become a truly personalized option for medical treatment. Although a
barrier to the widespread clinical application of these new therapies is their
financial cost, CRISPR technology has the potential to disrupt pathology
and cure genetic disease.
In terms of cost, projections range from 500,000 US dollars to $1.5
million. The cost could limit the availability of the technology to lower
socioeconomic groups, but with future advances, the cost is likely to fall.
To prepare the insurance industry, many leaders in the field are seeking to
address the challenges, working with payers, policy makers, and other key
experts in regulatory affairs and law to recommend payment programs
(Salzman et al. 2018). There have been various models proposed to handle
the high cost of gene therapy such as a value-based payment model which
would link payments to actual outcomes observed over an extended time
period. One possibility would be for the pharmaceutical company to
reimburse the insurance provider the cost of the treatment if, after three
years there is no clinical improvement. Visual science researchers and
clinicians continue to conduct ground breaking studies that will soon
translate into new therapeutic options.
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THE APPLICATION OF CRISPR/CAS9

C. B. Tara Moore1, , PhD, Larry A. DeDionisio2, , PhD,

Hila Roshanravan2, , PhD, Connie Chao-Shern2,

and M. Andrew Nesbit1, , PhD ‖

Clustered regularly interspaced short palindromic repeats (CRISPR)

2. APPROACHES FOR GENE EDITING WITH CRISPR/CAS

Figure 1. Cas9 programmed by tracrRNA:crRNA duplex.

Cas9 is a crucial component in the type II CRISPR/Cas system and

aureus Cas9, which recognises a 5–NNGRRT– 3’ PAM (Belfort and

2.1. Minimising off Target Cleavage and

There are limitations that can hinder applications of the CRISPR/Cas9

Table 1. Different techniques to identify off-targets

Several methods have been devised to directly identify cleaved sites in

nucleotide insertions and deletions, referred to as ‘indels’. This is

Table 2. Different strategies to enhance Cas9 specificity

highly similar off-target sites (Guilinger, Thompson, and Liu 2014,

2.2. Expanding the CRISPR/Cas Protospacer

Cas9 nucleases from various bacterial species have been shown to

Table 3. CRISPR/Cas Protospacer Adjacent Motif (PAM) repertoire

Source Nuclease type PAM sequence

3. CRISPR-CAS9 GENE EDITING IN GENETIC

millions of people worldwide and have an immense socioeconomic impact.

3.1. Applications for Retinal Eye Disease

Research into CRISPR/CAS9 gene editing as applied to ocular

congenital amaurosis 10 (LCA10) bearing the CEP290 splice mutation

3.2. Corneal Autosomal Dominant Genetic Eye

The majority of corneal dystrophies are the result of an autosomal

p.Leu132Pro which generates a novel PAM site was identified for

Table 4. Corneal dystrophies: associated inheritance pattern, gene

Corneal layer Disease Inheritance Genetic Gene Gene/s IC3D Category

Corneal layer Disease Inheritance Genetic Gene Gene/s IC3D Category

3.3. Mutant Allele Targeting, a Dual Cut

There are at least 70 known missense mutations located in the coding

to 10 kb coupled to precise NHEJ effectively knocked down a mutant

Figure 2. Guide-length screen to determine the effect on specificity of a guide-specific

While our study focussed on the issue of on-target allele-specificity in

CRISPR/Cas9 system resulted in a loss of CEC identity through the

3.4. Multiplexing in the CRISPR System for

Complex genetic ocular diseases like primary open angle glaucoma

A construct referred to as CRISPR-on was used in a study where an

Table 5. Current approaches for providing multiple gRNAs

Multiplex CRISPR System Description

networks (Nissim et al. 2014).

Multiplex CRISPR System Description

4. DELIVERY OF CRISPR/CAS TO THE EYE -

Figure 3. Delivery of Cas9-sgRNA complex. A. Viral vector based delivery B.

Efficient and effective delivery of the CRISPR/Cas machinery to the

4.1. In Vivo Systems

A range of viral systems including adenoviruses, AAVs and

intracameral injection of the AAV hybrid serotype vector, AAV-2/9 was

packaging restrictions, and they have lower immunogenicity and toxicity

editing could be best achieved by combining a viral coupled to non-viral

4.2. Ex Vivo Systems

The use of iPSCs in gene editing technologies hold great promise in

LESCs, and for those TGFBI mutations resulting in an intrastromal

Figure 4. CRISPR therapeutic approach; transition to clinics.

5. ANIMAL MODELS FOR EYE DISEASE

The generation of new model systems with genome-editing tools, in

CRISPR/Cas systems are also applied to mammalian models of the

6. RECENT PROGRESS IN CLINICAL GENE THERAPY

Research for transitioning effective gene therapy strategies for ocular