Low-passage HEK 293T cells were used for the production of AAV-containing cell lysates. Cells were seeded into six-well plates (CytoOne) at a density of 350,000 cells per well. The following day, cells were triple-transfected with (i) an AAV helper plasmid carrying AAV2 rep and cap genes, (ii) an adenoviral plasmid providing helper functions for AAV production, and (iii) the AAV vector plasmid using 1.33 μg of each construct and 8 μl of TurboFect Transfection Reagent (Thermo Fisher Scientific) per well. The AAV vector plasmid encoded either (i) a U6 promoter–driven sgRNA targeting the AAVS1 locus as well as an RSV promoter–driven GFP (used as transduction reporter), (ii) Cas9, or (iii) the AcrIIA4 variant (LOV2-AcrIIA4 Insertion 3 in table S2; Addgene no. 113036). Three days after transfection, cells were collected in 300 μl of phosphate-buffered saline (PBS) and subjected to five freeze-thaw cycles by alternating between snap-freezing in liquid nitrogen and 37°C in a water bath. Centrifugation at 18,000g was applied for 10 min to remove cell debris, and the supernatant containing AAV particles was stored at 4°C until use.

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