Mutation frequencies at on-target and potential off-target sites of the C4BPB- and CCR5-specific RGENs in K562 cells. (A,B) Cells were transfected with crRNA, tracrRNA, and the Cas9 plasmid or the Cas9 plasmid alone (negative control). PCR amplicons that span the on-target site and potential off-target sites were subjected to deep sequencing. Sequences that contained indels around the expected cleavage site were considered to be RGEN-induced mutations. Mismatched bases are shown in red. The PAM sequence is shown in blue. (C) In vitro cleavage assay of on-target or potential off-target sequences by the CCR5-specific RGEN. Plasmids that contain putative off-target (upper) or hybrid (middle) sequences were digested with the recombinant Cas9 protein complexed with crRNA and tracrRNA. Asterisks indicate cleaved DNA bands. (Bottom) DNA sequences of the on-target, off-target, and hybrid sites.
