Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa
- Department of Ophthalmology, Third Hospital, Peking University, China
- Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital,, China
- Beijing Chinagene Co., LTD, China
- Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, China
Figures
Figure 1
Schematic representation of allele-specific sgRNA design for RHO-T17M mutation.
(A) and confirmation of the specificity achieved using SaCas9 protein complexed with an sgRNA to target the mutant RHO sequence and its corresponding WT sequence in vitro (B), the full-length …
Figure 2 with 1 supplement
In vitro knockdown of human RHO-T17M expression.
(A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and …
Figure 2—figure supplement 1
The knockdown of human RHO-T17M gene expression in 293T cells stably expressing SaCas9 and SgRNA.
(A) SaCas9/SgRNA_GFP stable cell line generation of 293T and transfection of human RHO cDNA plasmid. The calculation of mCherry/GFP ratio indicated that SaCas9/SgRNA did not cut the WT RHO allele (B–…
Figure 3 with 1 supplement
Determination of specificity and cutting efficiency of SaCas9 and sgRNA in patient-specific iPSCs.
(A) Photograph of UCs. Scale bar = 100 μm. (B) Photograph of iPSCs. Scale bar = 200 μm. (C–D) Sanger sequencing results showed that P1 iPS had the heterozygous c.50C>T mutation. IF staining showed …
Figure 3—figure supplement 1
Hi-Tom sequence results of SaCas9/17-Sg2-treated P1 iPS (A) and P1 iPS colony (B).
Figure 4
AAV2/8-mediated SaCas9 and sgRNA expression in mouse retinas.
(A) (Top) AAV2/8 vectors expressing EGFP under the control of EFS promoter. (Bottom) AAV2/8 vectors expressing SaCas9 under the control of EFS promoter and sgRNA under the control of U6 promoter …
Figure 5 with 2 supplements
CRISPR/Cas9 targeting of c.50C>T RHO dominant variant encoding p.T17M in Mut-Rhowt/hum mouse retinas.
(A) T7E1 assay was performed on GFP+ and GFP- retinas to detect gene-editing activity of AAV-SaCas9/17-Sg2 in Mut-Rhowt/hum mouse retinas at 5×108, 1×109, and 3×109 doses, the full-length amplicon …
Figure 5—figure supplement 1
Analysis of gene-editing specificity and efficiency in RHO humanized mouse retinas.
(A) T7E1 assay performed on GFP+ and GFP- retinas indicated undetectable gene-editing activity (gene-editing specificity) of AAV-SaCas9/17-Sg2 in Rhowt/hum mouse at 5×108, 1×109, and 3×109 doses, …
Figure 5—figure supplement 2
Phenotypes of the humanized mouse models.
(A) Scotopic and photopic ERG results of Rhowt/wt and Mut-Rhowt/hum mice with increasing age. Error bars show SEM, and the significance was calculated using two-tailed paired t-test, *p<0.05; ***p<0.…
Figure 6 with 1 supplement
Expression of the mutant human RHO allele after gene editing with SaCas9/17-Sg2 in vitro.
(A) Schematic view of the different human RHO gene variants created by gene editing. (Top) Map of the pEGFPN1 vector used to overexpress these variants. (Bottom) The description of variants at DNA …
Figure 6—figure supplement 1
Expression of the mutant human RHO allele after gene editing with SaCas9/17Sg-2 in vitro.
(A–B) GFP expression in 293T cells transfected with pEGFPN1 vector carrying RHO-5m (five variants in RHO CDS) and four edited variants, 24 hr after transfection. Scale bar = 50 μm.
Figure 7
Expression of the RHO-5m allele after gene editing with AAV-based SaCas9/17-Sg2 in Rhohum/m-hum retinas.
(A) T7E1 assay was performed on GFP+ and GFP- retinas to detect cutting activity of AAV-SaCas9/17-Sg2 in Rhohum/m-hum retinas at 3 mpi, the full-length amplicon was 700 bp, the two truncated …
Figure 8 with 1 supplement
Significant improvement of retinal functions with AAV-based SaCas9/17-Sg2 in Mut-Rhowt/hum mice determined by full-field ERG.
Representative scotopic ERG (at the flash intensity of 10 cd*s/m2) waveforms of Mut-Rhowt/hum mice treated with AAV-SaCas9/17-Sg2 (blue) and AAV-SaCas9/CTRL (red) at 1×109 dose (3 mpi, A), 3×109 …
Figure 8—figure supplement 1
The ERG results of AAV-SaCas9/17-Sg12-treated Mut-Rhowt/hum mice (n=8), AAV-SaCas9/CTRL-treated mice (n=5), untreated Mut-Rhowt/hum mice (n=5), and untreated WT mice (n=7).
The mean amplitudes of scotopic a-wave (B) and b-wave (B) at 10 cd*s/m2. Error bars show SEM, and the significance was calculated using two-tailed unpaired t-test, *p<0.05; **p<0.01; ***p<0.005; …
Figure 9 with 4 supplements
Photoreceptor cell preservation after gene editing with AAV-based SaCas9/17-Sg2 in Mut-Rhowt/hum mice.
(A) At 9 mpi, IF staining of rhodopsin (4D2, red) and PNA (red) from age-matched untreated mouse retina, the AAV-SaCas9/CTRL-treated and AAV-SaCas9/17-Sg2-treated retinal regions (GFP+) in Mut-Rhowt/…
Figure 9—figure supplement 1
Retinal images after gene editing with AAV-based SaCas9/17-Sg2 in Mut-Rhowt/hum mice at 4 mpi.
Retinal images from the treated (GFP+) regions in Mut-Rhowt/hum mice at 1×109 dose (A) and 3×109 dose (C). (Upper) Representative image of mouse retina treated with AAV-SaCas9/CTRL. (Lower) …
Figure 9—figure supplement 2
Flat-mount images of Mut-Rhowt/hum retinas after treatment.
At 9 mpi, HE and IF staining of AAV-SaCas9/CTRL-treated (A) and AAV-SaCas9/17-Sg2-treated retinas in Rhowt/hum mice at 1×109 (B) and 3×109 dose (C).At 11 mpi, HE staining of AAV-SaCas9/CTRL-treated …
Figure 9—figure supplement 3
Retinal images after gene editing with AAV-based SaCas9/17-Sg2 in Mut-Rhowt/hum mice.
(A) Retinal images of untreated Mut-Rhowt/hum mice (P10.5m), mutant rhodopsin was retained in RIS and cell bodies (4D2, red). Retinal images from the treated (GFP+) regions in Mut-Rhowt/hum mice at …
Figure 9—figure supplement 4
Inner retinal cells preservation after gene editing with AAV-based SaCas9/17-Sg2 in Mut-Rhowt/hum mice.
At 9mpi (A) and 11 mpi (B), IF staining of PKC-α (red), calbindin (red), CRALBP (red) from age-matched untreated mouse retina, the AAV-SaCas9/CTRL-treated and AAV-SaCas9/17-Sg2-treated retinal …
Figure 10 with 2 supplements
Examination of SaCas9/17-Sg2 off-target effects in human gDNA using WGS.
Identification of SNVs (A) and indels (C) in 293T cells transfected with 17-Sg2 plasmid at the WGS level. The type of SNVs (B) and indels (D) in 293T cells transfected with 17-Sg2 plasmid and …
Figure 10—figure supplement 1
Analysis of SaCas9/17-Sg2 off-target activity.
T7E1 assay showed undetectable off-target activity for all candidate sites for 17-Sg2 (OT1-OT10).
Figure 10—figure supplement 2
Safety analysis of AAV-SaCas9/17-Sg2 in NHP.
(A) The qPCR result indicated that AAV-SaCas9/17-Sg2 could express in NHP retina. (B) Multifocal ERG results of untreated NHP and an AAV-SaCas9/17-Sg2-treated NHP. The multifocal ERG amplitudes of …
Additional files
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Supplementary file 1
Supplementary tables.
(a) Results of TA cloning and Sanger sequencing in 293T cells. (b) A comprehensive summary of mouse experiments. (c) Results of TA cloning and Sanger sequencing in AAV-based SaCas9/17-Sg2-treated Mut-Rhowt/hum retinas. (d) Off-target sites of 17-Sg2 obtained from Benchling (https://www.benchling.com/). (e) Off-target sites of 17-Sg2 obtained from Cas-OFFinder (http://www.rgenome.net/cas-offinder) and NGS results. (f) Off-target sites of 17-Sg2 obtained from Cas-OFFinder (http://www.rgenome.net/cas-offinder) and NGS results. (g) The number of off-target sites of 17-Sg2 obtained from Cas-OFFinder (http://www.rgenome.net/cas-offinder). (h) Whole-genome sequencing results of off-target activity for SaCas9/17Sg-2. (i) List of primers.
- https://cdn.elifesciences.org/articles/84065/elife-84065-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84065/elife-84065-mdarchecklist1-v2.pdf
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Source data 1
Source data files of the raw unedited gels or blots.
- https://cdn.elifesciences.org/articles/84065/elife-84065-data1-v2.zip
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Source data 2
Source data files of the gels or blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/84065/elife-84065-data2-v2.zip
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Source data 3
Source data files of NGS results and ERG statistical data.
- https://cdn.elifesciences.org/articles/84065/elife-84065-data3-v2.zip
