Vijaya Sree PPT 2 CDFD

• Retinitis pigmentosa (RP) is a group of genetically heterogeneous
retinal diseases.
• Three million people are affected across the globe with an incidence of
1 in 3,500 live births.
• Inherited autosomal dominant, recessive and X-linked manner
• More than 3,000 disease-associated variants in approximately 70
disease-causing genes have been causally associated with RP.
• Currently more than 150 documented missense/nonsense variants in the
rhodopsin (RHO) gene are associated with an autosomal dominant RP
(adRP) phenotype.
• The RHO gene encodes for rhodopsin (RHO) protein, a visual pigment
found in rod photoreceptors, responsible for converting photons into
chemical signals initiating vision.
• Rhodopsin is a 348 amino acid G-protein-coupled receptor,
chromophore 11-cis-retinal, and an intracellular C-terminal domain,
involved in vectorial transport of rhodopsin to rod outer segments
(OSs).
• High prevalence variant in c.68C>A RHO allele encoding the
p.Pro23His variant in the United States which have already been
reported.
• But the most common residue affected and neglected so far variant in
European individuals is c.1039C>T RHO allele encoding the
p.Pro347His variant.
CRISPR cas9:
• Cas9 nucleases they have used are Streptococcus pyogenes Cas9
(SpCas9) WT and the high-fidelity variant carrying seven amino acid
substitutions, Asn497Ala, Arg661Ala, Gln695Ala, Gln926Ala,
Asp1135Val, Arg1335Gln, and Thr1337Arg (hereafter referred to as
the VQRHF1)
• Allele specific gRNAs they have used is
• SpCas9_gRNA1
• VQRHF1-SpCas9_gRNA5
Methods:
• Site directed mutagenesis(plasmid selection,cell culturing)
• Lentiviral vector viral production
• Qpcr(vector copy number detection)
• Semiquantitative PCR and RT-PCR anlysis.
• Taregeted deep sequencing and off target analysis
• Immunoblotting analysis
• Immunoflouroscence and In-Cell western analysis
• Cytotoxicity
• Animal care(mice)
• Subretinal injection of AAV vectors in mice
• Electrophysiological recording and pupillary light response analysis
• Histological analysis through IHC
• Statistical analysis
(A) Schematic representation of human chromosome 3. The picture illustrates two gRNAs (gRNA1 and
gRNA5) targeting the variant (T, in bold) in the exon 5 of RHO and the PAM sequences. Capital letters
indicate the exon 5, whereas the 30 UTR is in lowercase.
(B) CRISPResso analysis of NGS data obtained on Pro347Ser and WT RHO HeLa clones transfected with effector
plasmids (SpCas9_gRNA1 in blue and VQRHF1-SpCas9_gRNA5 in orange). The experiment was performed in
triplicate, and the mean is presented. (C) CRISPResso analysis of indels occurring in c.1039C>T RHO transgene
and in the endogenous WT RHO gene after transfection of effector plasmids in Pro347Ser RHO HeLa clone. The
experiment was performed in triplicate and is presented as mean 5 SEM
(D) Indels analysis of off-target sites
predicted for gRNA1 (top) and for gRNA5
(bottom). The color bars (blue and orange)
represent values ascending from bottom
to top ranking the indels frequency
(E and F) CRISPResso graphic representation of indels
scored in the target site of Pro347Ser RHO HeLa cells
transfected with SpCas9_gRNA1 (E) and VQRHF1-
SpCas9_gRNA5 (F). The top sequence is the
unmodified reference. The percentage of indel
frequency and the number of reads scored are
indicated. Red boxes indicate nucleotide insertion
and nucleotide in bold indicates substitution.
(G) Type of indels, and their relative percentage, generated in Pro347Ser RHO HeLa clone transfected with
SpCas9_gRNA1 and VQRHF1- SpCas9_gRNA5.
(H) CRISPResso analysis of indels
generated by SpCas9_gRNA1 (top pie
chart) and VQRHF1-SpCas9_gRNA5
(bottom pie chart) leading to frameshift
or in-frame alterations.
(A) Immunofluorescence analysis of CHO cells transfected with plasmids coding for RHO variants. Permeabilized
cells (left) were stained with anti-BIP and anti-4D2 antibodies, and the scale bar represents 5 mm. Cells not
permeabilized (right) were stained with anti-4D2 and anti-HA antibodies, and the scale bar represents 10 mm.
(B) Immunoblot analysis of WT, p.Pro347Ser, and
most frequent RHO variants generated after editing
expressed in CHO cells transfected with the
respective coding plasmids. Anti-HA antibody was
used to detect rhodopsin (RHO). The
immunoblotting was normalized with an anti-beta-
actin antibody.
C) Densitometric quantification of RHO

protein level detected by western blot
(Figure 2B) in CHO cells transfected with
plasmids for the expression of RHO P347S,
WT or mutants. The experiment was
performed in duplicate and presented as
mean ± SEM. * p-value < 0.05, ** p-value <
0.01.
(C and D) Densitometric analysis of immunoblots performed on CHO cells transfected with plasmids
coding for p.Pro347Ser, del9, del12.1, and del12.5 RHO variants and treated with 10 mg/mL
cycloheximide (CHX) and 50 mM MG-132 proteasome inhibitor. The experiment was performed in
triplicate and is presented as mean 5 SEM. *p value < 0.05.
(A) Immunoblot for rhodopsin protein expressed in Pro347Ser and WT RHO HeLa clones transfected
with SpCas9_gRNA1 and VQRHF1- SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-
SpCas9). 4D2 antibody was used to detect rhodopsin. The immunoblotting was normalized with anti-
beta-actin antibody
(B) Densitometric quantification of
rhodopsin protein level normalized to
beta-actin after editing. The experiment
was performed in triplicate and is
presented as mean 5 SEM. *p < 0.05; **p
< 0.01.
(C) Pro347Ser and WT RHO HeLa clones were transfected with SpCas9_gRNA1 and VQRHF1-
SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-SpCas9). The relative quantity (RQ)
was calculated with the 2-DDCT quantification and is reported in the y axis. Each sample was run in
triplicate. **p < 0.01.
(D) Lactate dehydrogenase (LDH) assay of Pro347Ser RHO HeLa clone transfected with SpCas9_gRNA1 and
VQRHF1-SpCas9_gRNA5 plasmids and control plasmids (SpCas9 and VQRHF1-SpCas9). Mock-transfected
Pro347Ser RHO HeLa cells and WT RHO HeLa cells were used as positive and negative controls. The experiment
was performed in triplicate and is presented as mean 5 SEM. *p value < 0.05.
Allele-specific editing in mouse photoreceptors
(A) (Top) a scheme of the experimental

timeline is depicted; (middle) AAV2/8 vectors
expressing the WT or VQRHF1 SpCas9 under
the control of IRBP promoter are
schematized; (bottom) AAV2/8 vectors
expressing the gRNA (gRNA1, gRNA5, or
scramble) and the GFP under the control of
RHO promoter
(B) Indels frequency
determined by NGS in
retinae injected with
effector vectors.
(C) Representation of indels scored
in retinae treated with gRNA1 or
gRNA5 AAV vector coupled to
appropriate AAV-SpCas9 vector.
Nucleotides inserted or deleted are
reported on the right.
(D) Downregulation of RHO transcript coding for
p.Pro347Ser RHO in retinae injected with effector
vectors with respect to retinae treated with scramble
vectors. The averages are depicted with a bar. *p <
0.05.
(E) Rhodopsin localization was
investigated at P50 in retinae
injected with effector vectors
using 4D2 and 1D4 antibodies
against rhodopsin.
Representative images are
shown. Scale bar represents 10
mm. Zoomed areas of
photoreceptors are shown on
the side. OS, outer segment; IS,
inner segment; ONL, outer
nuclear layer.
Significant improvement of retinal electrical function and pupillary light response
(A and B) Pro347Ser transgenic mice injected with effector or control AAV2/8 vectors were examined at P40 by ERG
(A), as shown in the data point distribution of B-wave amplitude at 20 cd.s/m2 , and PLR analysis (B). Individual eyes
are depicted as squares. Data are presented as mean 5 SEM. *p < 0.05; **p < 0.01.
Conclusion:
• Retinitis pigmentosa (RP) is a group of progressive retinal
degenerations of mostly monogenic inheritance,
• Heterozygous variants in the rhodopsin (RHO) gene, missense variants
at C-terminal p.Pro347Ser, cause severe adRP recurrently in European
affected individuals.
• CRISPR/Cas9 to selectively target the p.Pro347Ser variant while
preserving the wild-type RHO allele in vitro and in a mouse model of
adRP.
• Detailed in vitro, genomic, and biochemical characterization of the
rhodopsin C-terminal editing shows a safe downregulation of
p.Pro347Ser expression leading to partial recovery of photoreceptor
function in a transgenic mouse model treated with adeno-associated
viral vectors.
• CRISPR/Cas9-mediated allele-specific editing paves the way for a
permanent and precise correction of heterozygous variants in
dominantly inherited retinal diseases.

Vijaya Sree PPT 2 CDFD

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• Retinitis pigmentosa (RP) is a group of genetically heterogeneous

C) Densitometric quantification of RHO

(A) (Top) a scheme of the experimental

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