T7 Endonuclease Assay for Genome Editing

The T7 endonuclease assay was performed as described previously [21 (link), 22 (link)]. Briefly, genomic DNA was isolated using DNeasy Blood and Tissue kits (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The region of DNA containing the nuclease target site was PCR-amplified using nested primers. Amplicons were denatured by heating and annealed to form heteroduplex DNA, which was then treated with 5 units of T7E1 (New England Biolabs, MA, USA) for 15 to 20 min at 37 °C, followed by 2% agarose gel electrophoresis. Mutation frequencies were calculated based on band intensity using ImageJ software and the following equation: mutation frequency (%) = 100 × (1 - [1 - fraction cleaved]^1/2), where the fraction cleaved was the total relative density of the cleavage bands divided by the sum of the relative density of the cleaved and uncut bands. The oligonucleotide sequences used to obtain the PCR amplicons from the on-target genes for the T7E1 assay are listed in Supplementary Table S2. The oligonucleotide sequences used to obtain the PCR amplicons from the off-target genes for the T7E1 assay is previously described in [20 (link)]. The amplicon sizes of the USP3 and their expected cleavage sizes after the T7E1 assay are summarized in Supplementary Table S3.