Fig. 1.

Structure of spCas9/sgRNA/DNA complex. a Overall structure of spCas9/sgRNA/DNA complex, with PI, HNH and RuvC domains shown in yellow, green and blue respectively. b and c Detailed structure showing the interaction between PI domain and PAM (b), with key amino acids highlighted in pink (c). d Detailed structure showing the HNH domain, RuvC domain and target DNA, with key amino acids (D10 and H840) shown in sphere. Mutation of Asparticacid 10 (D10) or Histidine 10 (H840) to Alanine (A) silences the RuvC or the HNH domain respectively, resulting in TS nickase or NTS nickase respectively. Mutation of both amino acids silences both domains, resulting in catalytic dead Cas9. e Detailed structure showing the key amino acids interaction with target strand in HNH domain. f Detailed structure showing the key amino acids interaction with target strand in RuvC domain