Fig 1: In vivo assessment of the on-target site.a Breakup of sequencing results of the on-target site in the genome-edited clones amplified from the retina collected 1 M or 3 M post-injection. MMEJ, NoMHA, and NoTS represents injection of protype MMEJ vector, MMEJ vector without microhomology arms, and MMEJ vector without gRNA target sites, respectively. HITI represents homology-independent targeted integration. See Supplementary Fig. 3 for vector map. Total clones sequenced were 57, 70, 67, 64 and 86 for MMEJ (1 M), MMEJ (3 M), NoMHA (1 M), NoTS (1 M), and HITI (1 M), respectively. Success indicates successful mutation replacement. Cleavage site indel represents indels in either gRNA cleavage site without replacement of IRD2 mutation (see Online Methods for detail). Note, co-existing mutation replacement and cleavage site indels, which is expected to occur as a consequence of repeated cleavage at the gRNA site was not observed. b Total editing rate. Percentage of clones that showed any sign of genome editing among the total clones analyzed. c Absolute success rate in the rods, assuming the cells comprise 75% of retinal neurons. d Estimation of detection efficiency of Success allele by subcloning and PCR. Observed % Success (vertical axis) were obtained by distinguishing the identity of the clones derived from PCR products amplified from mixture of Success and unedited mutant DNA templates at various ratio (horizontal axis). Total clones sequenced in this experiment were 61, 52, 64 and 61 for 5%, 10%, 20%, and 50% mixtures (rate of Success DNA versus total DNA), respectively. Intercept = −0.154, slope = 0.528, r2 = 0.99. e Estimated absolute success rate in the rods corrected for by the detection efficiency of Success alleles relative to unedited mutant allele, assuming 75% of retinal neurons are the rods. f RT-PCR of Gnat1 at 1 M (N = 4) and 3 M (N = 3). g RT-PCR of SaCas9 at 1 M (N = 4) and 3 M (N = 3) post-injection. h. RT-PCR of gRNA at 1 M (N = 4), and 3 M (N = 3) post-injection. Data represent the mean ± S.E.M. Source data are provided as a Source Data file.
Fig 2: In vivo characterization of mutation replacement genome editing.a Illustration of MMEJ-mediated mutation replacement. Genome of interest (GOI) with and without the mutation are excised at the flanking gRNA target sites (gRNA-T1 and -T2; dotted line) from mouse genome and AAV vector, respectively, by SaCas9 and two gRNAs. GOI without mutation is inserted into the genome using microhomology arms (MHA), thereby correcting the mutation. b GNAT1 staining. GNAT1-positive photoreceptors (arrowhead) were observed (section, left; flatmount, right). c Co-localization of Kusabira Orange (mKO1, red) probing SaCas9 expression and GNAT immunopositivity (inset, green). Scattered GNAT-positive cells were observed only in the area transduced with mKO1 (section, top; flatmount, bottom). Note, oversized reporter vector (5201 bp) drastically reduced editing efficiency. N = 4 d PNA and recoverin staining with quantification (N = 4). e RT-PCR of Rho, Pde6b, Rcvrn, and Pkcα (relative to Pde6ccpfl1/cpfl1 mice; N = 4 for all). f Rescue efficiency by RT-PCR of Gnat1 (relative to Pde6ccpfl1/cpfl1 mice; N = 4 for all). g 6-Hz flicker ERGs. N = 9, 9, 4, 4, and 4 for No treatment (NoTx), MMEJ, MMEJ + L-AP4, NoMHA, NoTS, respectively. In MMEJ + L-AP4, MMEJ vector and L-AP4 were sequentially injected. Amplitudes (−1.0 log.cd.s.m−2) relative to those of Pde6ccpfl1/cpfl1 mice indicate %rescue efficiency (bottom right). h. Single flash ERGs. The same group of mice used in g. Scale bar: 20 µm; Data represent mean ± S.E.M.; *P < 0.05 (Student’s t-test); ns, not significant; PAM, protospacer adjacent motif, OHS, over-hanging sequence; Abx, antibodies; NoTS, no gRNA target sites. Source data are provided as a Source Data file.
Fig 3: In vivo mutation replacement genome editing in a mouse model of retinal degeneration.a GNAT1-positive photoreceptors (arrowhead) following treatment of Gnat1IRD2/IRD2 mice shown in a retinal section (top) and a flatmount (bottom). Scale bar: 20 µm. b fVEPs recorded from contralateral visual cortices in treated and untreated eyes of the same mice. (N = 7). c Fear conditioning test, showing freezing time before (Baseline) and during (Stimulus) presentation of fear-conditioned light cue. Treated (Tx, N = 7) and untreated (NoTx, N = 6) Gnat1IRD2/IRD2 mice and CL57B6 mice (B6, N = 6). Data represent the mean ± S.E.M.; *P < 0.05 (Student’s t-test); ONL, outer nuclear layer. ns, not significant. Source data are provided as a Source Data file.
Fig 4: PrPSc accumulates first in cone photoreceptor inner segments. a At 67 dpi, rare small punctate PrPSc deposits (arrow) are present on cone photoreceptors marked with Cone Arrestin (green). Magenta in outer segment is autofluorescence of rhodopsin. b Cone opsin (white) marks cone outer segments with PrPSc (magenta) deposits associated mainly with cone inner segments (yellow arrow) at 104 dpi. The transition from faint to intense cone opsin staining marks the boundary between cone IS and cone OS (blue arrow). c Another cone-specific outer segment protein GNAT2 (green) shows obvious connection with PrPSc (magenta) staining cone inner segments at 104 dpi (arrows). d At 104 dpi, rod-specific marker, GNAT1 (green) stains rod inner and outer segments, but spares cones (arrowheads). PrPSc (magenta) accumulations are present in the dark GNAT1-free areas (arrows). e A separate experiment done with 22L scrapie strain shows same association of PrPSc with cone inner segments at 123 dpi, suggesting cone specificity is not strain-specific. Scale bar = 5 µm. a, b, d are confocal z-stacks, c, e are widefield images
Fig 5: Visual restoration by in vivo mutation-replacement genome editing.Flash visually evoked potentials (fVEP) of the visual cortex contralateral to the eyes in response to flashes of various intensities. MMEJ indicates eyes treated with Gnat1 mutation replacement (N = 9) and OE (over-expression) indicates those with Gnat1 gene supplementation (N = 6), both delivered by single AAV. NoTx refers to untreated eyes (N = 9). Control Pde6ccpfl1/cpfl1 mice (N = 5). Note, light sensitivity as defined in the Methods was increased by ~4 log unit after MMEJ-mediated genome editing, which was not significantly different to the effect mediated by OE (right lower panel). b Pattern VEPs. N = 11, 10, and 6 for MMEJ, untreated and OE, respectively. c Fear conditioning test. Freezing time before (Baseline) and during (Stimulus) presentation of fear-conditioned light cue from MMEJ treated (N = 9) and untreated (N = 6) mice. d Optokinetic response. Note threshold of spatial resolution of vision (visual acuity) was not different in the MMEJ and OE. N = 10, 7, and 4 for MMEJ, OE, and NoTx, respectively. Control Pde6ccpfl1/cpfl1 mice (N = 6). Data represent the mean ± S.E.M.; *P < 0.05 (a, b, d, ANOVAs followed by Tukey’s post hoc test; c Student’s t-test); nd, non-detectable; ns, not significant. Source data are provided as a Source Data file.
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