Fig. 5.

Double-nicking induced efficient genome editing with lower off-target effects. a Two sgRNA targeting sites in sox9. Letters in blue is the target site 1 while letters in red is the target site 2. The red arrows point the predicted cleavage sites, and the PAMs are underlined. b Sequencing data of the indels induced by D10A and the sgRNA pairs. Letters in bold is the wild type sequence, red dashes indicate deletions. c T7E1 assay of genome editing induce by wide-type Cas9 or by D10A. d Quantification of the T7E1 assay shown in (c). e Mutagenesis at a representative off-target locus (sox9-600) induced by Cas9 and sox9 T1gRNA or by D10A and sox9 T1 and T2 sgRNAs. PCR was performed to amplify the targeted sox9-600 locus. Amplicons harboring targeted gene fragments were TA-cloned into pMD-18T. The indel mutagenesis rates were calculated by direct sequencing. (f) Comparison of the off-target efficiency induced by D10A with double nicking versus wide type Cas9 with sox9 sgRNA