Sex as a biological variable. Our study examined male and female animals, and similar findings were reported for both sexes.

Animals. Pde6anmf363/nmf363 mice (29) were a gift from Vinit B. Mahajan (Department of Ophthalmology, Stanford University). Animals were housed under a 12-hour light/12-hour dark cycle with access to water and food.

Plasmid construction. The AAV-SaCas9 plasmid was obtained from Addgene (plasmid 61591). The sgRNAs targeting Nrl/Nr2e3 were designed by Cas-Designer (47). All sgRNA oligos were synthesized by Azenta Life Sciences and then annealed and ligated into the BsaI-digested AAV-SaCas9 plasmid. The sequences of sgRNA oligos are listed in the Supplemental Table 1.

Cell culture and transfection. The N2a cell line (ATCC, CCL-131) was cultured in Dulbecco’s Modified Eagle’s Medium (Corning, 10013CV) supplemented with 10% fetal bovine serum and incubated at 37°C in an atmosphere of 5% CO2. The cells were seeded in 24-well plates and transfected using PolyJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, SL100688) according to the manufacturer’s instructions. Briefly, 1.5 μL PolyJet reagent with 500 ng AAV-SaCas9 plasmid was added to each well. After 72 hours, the transfected cells were lysed using the One Step Mouse Genotyping Kit (Vazyme, PD101) according to the manufacturer’s instructions. The primers used to amplify target sequences are listed in Supplemental Table 3. Sanger sequencing results were analyzed by TIDE (48).

AAV production and injection. The AAV-SaCas9–Nrl-sg2 was packaged with serotype AAV2.NN (30) and generated by the AAVnerGene. The titer of the produced AAV was 2 × 1013 genome copies/mL (GC/mL). For AAV delivery, Pde6a mice received approximately 1 × 1010 GC AAV per eye after dilution via subretinal injection at P7. Mice were anesthetized with ketamine, and pupils were dilated by 1% topical tropicamide. Subretinal injections were administered under an ophthalmic surgical microscope with Picospritzer III microinjection system and a custom-crafted glass micropipette. Approximately 0.5 μL AAV was injected into the subretinal space through a small scleral incision.

Targeted deep DNA sequencing. Top 10 potential off-target sites for Nrl-sg2 were predicted using Cas-OFFinder (49). Genomic DNA was extracted from injected mouse retinas at P60 using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, DC102), according to the manufacturer’s protocols. Deep-sequencing primers were designed with generic adapters, and PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, F530L). Targeted deep DNA sequencing was conducted using the Amplicon-EZ sequencing service from Azenta Life Sciences. More than 50,000 reads were generated with each sample using the Illumina platform. Data analysis was performed with CRISPResso2 (50). The primers used to amplify on-target and off-target sequences are listed in Supplemental Tables 3 and 4.

Western blot analysis. For Western blot analysis, the mouse retinas were dissected and homogenized in 200 μL RIPA Lysis Buffer (MilliporeSigma, 20-188) supplemented with a protease inhibitor cocktail (Thermo Scientific, 78430). The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, 23227). Anti-NRL antibody (Proteintech, 17388-1-AP, 1:500) and anti-Alpha Tubulin antibody (Proteintech, 11224-1-AP, 1:5000) were used as primary antibody and internal control, respectively. Signals were acquired by direct measurement of chemiluminescence using a digital camera (Amersham Imager 600).

qPCR analysis. Total RNA was extracted from the mouse retinas using the Quick-RNA Miniprep Plus Kit (Zymo Research, R1058) according to the manufacturer’s instructions. The cDNA was synthesized with the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R212). Primers used for qPCR are listed in Supplemental Table 5. The qPCR was performed using the BioEasy SYBR Green I real-time PCR kit with the Bio-Rad CFX Opus 384 multicolor real-time PCR detection system. The relative gene expression normalized to Gapdh was determined by the 2–ΔΔCT method. All gene expression data experiments were performed 3 times, and data are expressed as the mean ± SEM.

Immunofluorescence analysis. Mice were euthanized using CO2, and eyeballs were enucleated and fixed in 4% PFA. Retinas were carefully dissected and subjected to a sucrose gradient series (5%, 15%, 30% sucrose). The retinas were then embedded in OCT compound and stored at –80°C. Cryosections of 15 mm thickness were prepared using a Leica CM1950 cryostat (Leica Biosystems). The retinal cryosections were rinsed in PBS, blocked in a solution comprising 0.1% Triton X-100 and 3% BSA in PBS for 30 minutes at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in the blocking buffer within a humidified chamber. Following 3 PBS washes with 0.1% Triton X-100, sections were exposed to secondary antibodies for 2 hours. DAPI was used to counterstain cell nuclei for 10 minutes. Slides were then mounted using Fluoromount-G mounting medium (Southern Biotech) and covered with a coverslip. The following antibodies were used: rabbit anti-HA tag (Cell Signaling, 3724, 1:500), mouse anti-Rhodopsin (Abcam, ab5417, 1:500), rabbit anti-Cone arrestin (Millipore, AB15282, 1:500), rabbit anti–S-opsin (Millipore, AB5407, 1:500), and rabbit anti-M-opsin (Millipore, AB5405, 1:500). The Alexa Fluor 555–conjugated anti-mouse or rabbit IgG (Invitrogen, 1:500) was used as a secondary antibody. All images of retinal sections were captured by a Zeiss LSM880 inverted confocal microscope. The fluorescence intensities were quantified by ImageJ software (NIH).

ERG. Mice were given 12 hours to adapt to the dark before ERG recordings were taken, during which they were anesthetized by ketamine based on their body weight (0.08 mg ketamine/g + 0.01 mg xylazine), and their pupils were dilated by 1% tropicamide. The ERG was performed with an ERG stimulator (Celeris, Diagnosys LLC) according to the manufacturer’s instructions. For scotopic ERG, mice were stimulated with flashes of 0.01, 0.1, and 1 cd.s/m2 light intensity. For photopic ERG, mice were given 10 minutes to adapt to the light and then stimulated with flashes of 1, 3, and 10 cd.s/m2 light intensity.

OKR. The detailed procedure has been previously published (51, 52). Briefly, the OKR) was assessed using the OptoMotry system (CerebralMechanics Inc.), a virtual-reality platform designed to swiftly quantify visuomotor behavior. Mice were positioned on a central platform surrounded by 4 computer monitors equipped with a video camera positioned overhead to record the animal’s movements. A rotating cylinder displaying vertical sine-wave gratings was projected onto the monitors. The OptoMotry software controlled the spatial frequency of the grating to assess the spatial acuity (cycle/degree) of the mouse being tested. The mouse’s tracking of the gratings was reflected through head and neck movements. The maximum spatial frequency of each eye was determined by gradually increasing the spatial frequency of the grating until the mouse ceased tracking.

Statistics. All data are expressed as mean ± SEM of at least 3 individual determinations for all experiments. Data were analyzed by 2-tailed Student’s t test via GraphPad prism software 8.0.1. A probability value smaller than 0.05 (P < 0.05) was considered as statistically significant.

Study approval. All animal experimental procedures were performed in compliance with animal protocols approved by the IACUC at Stanford University School of Medicine (protocol ID 32223).

Data availability. Deep sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1121624). Values for all data points in graphs are reported in the a data values file.