Upon passaging the transfected cells, genomic DNA was isolated using the PureLink genomic DNA Mini Kit (Thermo Fisher Scientific). PCR primers for NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA) were designed with SnapGene 4.1 software (GSL Biotech LLC, IL, USA).

Primers for Alpl target region harboring CRISPR-target sites are as follows: (forward) 5′-actaaagggaacaaaagctgcttgggaatggctcctggttt-3′ and (reverse) 5′-actcactatagggcgaattgtgtcctcctgttgcgatgtgtga-3′. Primers for pBluescript KS (+) vector are as follows: (forward) 5′-aaccaggagccattcccaagcagcttttgttccctttagtgaggg-3′ and (reverse) 5′-cacatcgcaacaggaggacacaattcgccctatagtgagtcgt-3′.

PCR-amplified Alpl target region, PCR-linearized vector, and NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs) were mixed and incubated at 50°C for 15 min. The mixture was then transformed into NEB5α competent cells (New England BioLabs). The purified DNA from each clone was subject to sequencing analysis.

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