Extended Data Fig. 5: Biochemical properties of CbCas9.
From: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity
a, Left, in vitro cleavage of dsDNA targets containing different PAMs by CbCas9 at 15 min revealed by denaturing PAGE, S denotes substrates and P means cleavage products. Right, bar plot illustrating the cleavage efficiency of CbCas9 on each dsDNA target (PAMs are shown on the X-axis). Gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). b, Cleavage site mapping of SpCas9 (left) and CbCas9 (right) on the NTS or TS revealed by denaturing PAGE. S means substrates, PNTS means cleavage products of non-target strand and PTS means cleavage products of target strand. Gel image is representative of four independent experiments with similar results (n = 4 independent replicates). DNA markers of 28-nt, 30-nt, 32-nt and 34-nt were labelled. c, Model depicting cleavage sites for NTS and TS by SpCas9 and CbCas9 (marked in red arrows, PAM in blue). d, Schematic representation of DNA target (6-nt complementary) recognition by CbCas9. The hydrogen bonds of K1365 with N7 of dA3 on NTS, and Q1397 with O4 of dT(−4) on TS are coloured in red. The interactions with DNA backbone are coloured in blue. e, Left, In vitro cleavage of NTS and TS by CbCas9 and β-REC2-mutated CbCas9 revealed by denaturing PAGE; aliquots were collected with time points: 0, 2, 5, 15, 30, 60, 90, and 120 min (n = 3, each). Right, in vitro NTS and TS cleavage efficiency plot of CbCas9 and β-REC2-mutated CbCas9 on dsDNA substrates. Gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). The rate constant k values for CbCas9 in NTS and TS are 0.2053 and 0.3100; for β-REC2-mutated CbCas9, the corresponding k values are 0.055 and 0.047, respectively). For gel source data, see Supplementary Fig. 1.
