Extended Data Fig. 8: PcrIIC1 enhances CbCas9 interference activity on mismatched or long dsDNA targets.
From: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity
a, Left, model depicting in vitro evaluation of the mismatch tolerance on the protospacer region by CbCas9 and CbCas9–PcrIIC1 effectors. Right, bar chart illustrating the mismatch tolerance ratio of CbCas9 and CbCas9–PcrIIC1 effectors at different positions. The mismatch tolerance ratio of 0 means that the cleavage products are perfectly paired with the sgRNA at that position, and ratio of 1 means 25% paired (randomly distribution; see Methods). Data are presented as mean ± s.d. from three independent experiments. Statistical analysis was calculated using two-sided test, with significance determined by t-test. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. b, In vitro cleavage assay of target dsDNA containing ACAAC PAM with different lengths (28, 62, 300 and 600 bp) revealed by denaturing PAGE (28- and 62-bp dsDNA targets) and agarose gel (300- and 600-bp dsDNA targets); aliquots were collected with time points: 0, 2, 5, 15, 30, 60, 90, and 120 min. Gels shown are representative of three independent experiments with similar results (n = 3 independent replicates). For gel source data, see Supplementary Fig. 1. c, Cleavage efficiency plot of target dsDNA with different lengths by CbCas9 and CbCas9–PcrIIC1 effectors. Data are presented as mean ± s.d. (n = 3 independent replicates). d, Bar chart showing cleavage efficiency of dsDNA targets with different lengths at 120 min by CbCas9 and CbCas9–PcrIIC1 effectors. Data are presented as mean ± s.d. (n = 3 independent replicates). Statistical analysis was calculated using two-way ANOVA with multiple comparisons; ns, P > 0.1234; ****P < 0.0001. e, Equilibrium dissociation constant (Kd) of PcrIIC1 with CbCas9–sgRNA–dsDNA(62bp) was evaluated using MST, with 25 nM Red-tris-NTA labelled CbCas9 and varying concentrations of PcrIIC1. Data are presented as mean ± s.d. from three independent experiments with consistent results. Graphs are represented as FNorm [‰] against PcrIIC1 concentration. dCbCas9 indicates deactivated CbCas9 (D9A, H837A).
