After deep anesthesia, AAV9-SpCas9 and AAV9-TS4 sgRNA-Rpe65-donor (a total of 2 × 1010 or 2 × 1011 vg in 1 μl of phosphate-buffered saline) were mixed and injected into the subretinal space of the mouse eye through the vitreous cavity using a customized Nanofil syringe with a 33-gauge blunt needle (World Precision Instrument) under an operating microscope (Leica), as previously described (35).

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