T7 endonuclease I assay for genome modification
293T Cells were collected after 48 h post-transfection for genomic DNA
extraction. The genomic region flanking the target site of each gene was
PCR-amplified, and products were purified using DNA Clean Kit following
the manufacturer’s protocol. A total of ~ 200 ng
purified PCR amplicons was mixed with 1 μl NEBuffer 2 and diluted in
ddH2O to 10 μl, then subjected to a re-annealing process to form a
heteroduplex according to the reported procedure [44]. After
re-annealing, the products were treated with T7EI following recommending
protocol, and 2.5% agarose gels were used for further analysis. Indels
were calculated via band intensities based on previously reported method
[45].