Sequences of the elongation factor 1a short (EFS) promoter and human optimized SpCas9 with a nuclear localization signal, hemagglutinin (HA) tag, and bovine growth hormone poly-A tail were synthesized and subcloned into an AAV2 inverted terminal repeat (ITR)–based plasmid using Not I restriction sites. For the other AAV vector, sequences of the U6 promoter, TS4 sgRNA, and Rpe65 donor were also synthesized and subcloned into the AAV2 ITR–based plasmid using the same restriction sites. In the Rpe65 donor sequences, five nucleotides were substituted without causing codon changes to prevent possible TS4 sgRNA–mediated cleavage and to precisely distinguish knocked-in sequences from endogenous genomic sequences. Recombinant AAV particles were produced using a helper adenovirus-free packaging system. Briefly, HEK293 cells were cotransfected with pAAV-ITR-EFS-SpCas9 or pAAV-ITR-TS4rd12-sgRNA-Rpe65-donor, AAV9-capsid plasmid, and helper plasmid. After 3 days of transfection, the cells were lysed and the virus particles were purified by iodixanol gradient ultracentrifugation and concentrated to obtain titers greater than 1013 vg/ml.

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