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Analysis of Cas9-independent editing at the genome

WY Wenxia Yu
JL Jianan Li
SH Shisheng Huang
XL Xiangyang Li
PL Ping Li
GL Guanglei Li
AL Aibin Liang
TC Tian Chi
XH Xingxu Huang
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Orthogonal R-loop assay was performed as described [15]. Briefly, to check off-target editing at site 5, plasmids expressing the dSaCas9 (300 ng) and its gRNA targeting site 5 (200 ng) were co-transfected into HEK293T cells with plasmids expressing an indicated editor (300 ng) and its gRNA targeting EMX1-2 (200 ng). Cells were treated with puromycin, and the editing at both site 5 and EMX1-2 detected by Sanger sequencing as described in the previous section. Off-targeting at site 6 was examined in the same way, except that the plasmid expressing the sgRNA for site 6 was used instead of the site 5 gRNA.

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