At 6 weeks after AAV-mediated delivery of SpCas9 and TS4 sgRNA-Rpe65-donor, paraffin blocks were prepared from enucleated eyes. Thin sections were immunostained with anti-HA antibody (1:1,000; cat. no. 3F10, Roche) and anti-Rpe65 antibody (1:1,000; cat. no. sc-73616, Santa Cruz Biotechnologies), followed by treatment with Alexa Fluor 488 or 594 immunoglobulin G (IgG; 1:500; Thermo Fisher). Nuclear staining was performed using 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). The slides then were observed under a fluorescence microscope (Leica).

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