Detection of off-target sites in the AAVS1 site

To examine whether there were off-target effects in the 293T cell genome, we identified other potential off-target sgRNA sequences for Cas9 [27]. These off-target sequences all end before the NGG base and have the best match with the guide RNA. Subsequently, we selected the six high-probability off-target sites as shown in Figure S2D. However, no mutated indel sequences were detected upon alignment with NCBI reference sequences, indicating no off-target effects in this cell line.为探究293T细胞基因组中是否存在脱靶效应，我们针对Cas9鉴定出了其余潜在的脱靶单向导RNA（single guide RNA，sgRNA）序列[27]。此类脱靶序列均终止于NGG碱基位点之前，且与向导RNA（guide RNA）的匹配度最高。随后，我们选取了如图S2D所示的6个高概率脱靶位点。然而，通过与NCBI参考序列进行比对后未检测到突变的插入缺失（insertion-deletion，indel）序列，表明该细胞系未出现脱靶效应。