T7 endonuclease I (T7 EI) assay

Cells were seeded in 96-well plates and transfected using either the Neon Transfection System (Life Technologies) or the FuGENE HD Transfection Reagent (Promega) according to the manufacturer’s instructions. Genomic DNA was extracted 3 days after transfection for PCR. PCRs were performed across on- or off-target sites with site-specific primers (Supplementary Table 2). PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and then quantified. The T7 endonuclease I assay was carried out according to the manufacturer’s instructions (NEB, M0302). In brief, 200 ng of amplified PCR products were reannealed to form heteroduplex in 1X NEBuffer 2 using the following program: denaturation at 95 °C for 5 min, reannealing from 95 °C to 85 °C at −2 °C s⁻¹, hold at 85 °C for 1 min, cooling from 85 °C to 25 °C at −0.1 °C s⁻¹, and hold at 25 °C for 1 min, followed by cooling down to 4 °C. The samples were then subjected to T7 EI at 37 °C for 15 min and analysed on agarose gels. The gels were imaged and quantified by Geldoc XR+ system (Biorad). Indel occurrence was calculated using the following formula as described⁵: indel (%) = 100 × (1−(1 − f_cut)^1/2). f_cut is the fraction of the cleaved PCR product.