CRISPR/Cas9 Genome Editing

Tool
Potential applications in Ophthalmologic Genome Surgery
CRISPR/cas9 Genome Editing
Tool
CRISPR (clustered regularly
Cas9 ("CRISPR-associated
interspaced short palindromic
repeats)
DNA sequences found within the
genomes of prokaryotic organisms
(bacterias),
derived from DNA fragments from
viruses that have previously infected
the prokaryote,
used to detect and destroy DNA from
similar viruses during subsequent
infections,
play a key role in the antiviral
defense system of prokaryotes.
protein 9”)
enzyme that uses CRISPR
sequences as a guide to
recognize and cleave specific
strands of DNA that are
complementary to the CRISPR
sequence.
prokaryotic immune system
that confers resistance to
foreign genetic elements such
as those present within
plasmids and phages
Potential applications in
Ophthalmologic Genome Surgery

CRISPR/Cas9 Genome Editing Tool

CRISPR-Cas Genome Surgery in
Animal Models of the Eye
• Zebrafish Models of Human Eye Development (embriology
study)

• CRISPR-Cas Editing in Mammalian Models of Human Eye

Development (retinitis pigmentosa - to correct a nonsense
mutation, study of proliferation and migration of photoceptor cells
in vitro, Leber’s congenital amaurosis - retinal pigment
epithelium potassium channel mutation correction)

• CRISPR-Cas Therapeutic Tool Development in Mammalian

Models (retinitis pigmentosa, VEGF gene modulation -
neovascularization, age-related macular degeneration -
regeneration of protein structures
CRISPR-Cas Genome Surgery in
Human Eye Tissue
to knockout the PAX6 gene in human corneal epithelial cells (CEC) in
vitro - corneal pathologies (in vitro study)

proliferative vitreoretinopathy (produces complications during

retinal detachment) - knockdown of the b1,6 N-acetylglucosa-
minyltransferase V (MGAT) gene in RPE cells. (in vitro study)

to suppress VEGF-A secretion from human retinal pigment epithelial

(RPE) cells and angiogenesis. (in vitro study)

targeting the VEGF-R2 receptor also reduces neovascularization both

in vitro and in vivo
CRISPR-Cas Genome Surgery in
Human Eye Tissue
SNPs - whole exome sequencing.

Retinal detachment and vitreoretinopathy - In human

primary retinal pigment epithelial (hPRPE) cells, Mdm2 (murine
double minute 2) gene/tumor suppressor protein, p53. (in vitro
study)

selectively ablate the rhodopsin gene mutations S334ter and

P23H in rodent models of autosomal dominant retinitis
pigmentosa. (in vitro study)
CRISPR-Cas Genome Surgery in
Human Eye Tissue
Modification of Human Stem Cells for the Treatment of Eye
Disease

(Deriving stem cells from patients offers specific advantages for

testing targeted therapeutics and autologous
transplantation with reduced risk of rejection)- Induced
pluripotent stem cells (iPSCs)

E.g. Mutations that cause X-linked retinitis pigmentosa have

also been corrected in patient-derived induced pluripotent stem
cells. (in vitro study)
Limitations of
CRISPR Systems
• not very efficient and is somewhat
random.
• the frequency of DNA cleavage
can be variable.
• some indel mutations may be
silent,
• not be a viable strategy for
photoreceptors and RPE cells that
are postmitotic.

• off-target mutations
• Ethical issues
“What the eyes see and the ears hear, the mind believes.”

–Harry Houdini