Fig. 6

Cas9 can generate staggered DNA breaks. a–c Biochemical mapping identified non-canonical Cas9 cleavage sites. a Schematic of Cas9 cleavage assay. ‘cct’ is PAM. b In vitro cleavage of BamHI linearized or circular DNA by Cas9 protein and purified sgRNA. The expected cleavage products are 5 + 1 kb for linearizd iGFP plasmid. The size shift (arrowhead) of circular iGFP plasmid indicates Cas9 cleavage. c Sequencing analysis of cleaved products. Red arrowheads indicate Cas9 cleavages sites on two DNA strands. The 3′ terminal A or T (asterisks), caused by artifacts of sequencing reactions, indicate termination of primer extension and the position of the Cas9 cleavage sites [7]. The circled ‘G’ in sequencing trace indicates that Cas9 can cut at fourth nt on the non-complementary strand. The downstream weak ‘G’ peak overlapping with ‘A’ implies fifth nt cleavage. d Fourth nucleotide insertion of ‘C’ nucleotide* (+C) was frequently observed at sgPten target site after single sgRNA transfection in mouse cells. Indel representation is the ratio of selected indel versus all observed indels. Arrowhead indicates predicted Cas9 target sites. The position of the most abundant insertion (red arrow) is indicated in the target sequence. PAM sequence is in blue