Extended Data Fig. 7: PcrIIC1 enhances the DNA-binding capability, PAM tolerance and target cleavage efficiency of CbCas9 by interacting with the CTH domain.
From: Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity
a, Top, model of in vivo bacterial genome interference assay for CbCas9 and PcrIIC1. Bottom, bacteria genome interference assay revealed by bacterial culture plate with a series of 10-fold dilution gradients. CbCas9 and CbCas9–PcrIIC1 groups with non-target spacer and target spacers are denoted as Cb and Cb+P separately (Different targeting PAMs are shown above the plate). Data are representative of three independent experiments with similar results (n = 3 biologically independent replicates). b, Efficiency plot of the DNA-binding capability of CbCas9 and CbCas9–PcrIIC1 on 62-bp dsDNA target with different PAM (ACAAA and ACAAC) evaluated by EMSA. Data are presented as mean ± s.d. from three independent experiments. dCb and dCb+P indicate deactivated CbCas9 (D9A, H837A) and deactivated CbCas9–PcrIIC1 effectors, respectively. c, Top, in vitro cleavage of dsDNA targets with different PAMs by CbCas9 and CbCas9–PcrIIC1 at 15 min, revealed by denaturing PAGE. S denotes substrates. Bottom, bar plot of cleavage efficiency (different PAMs are shown on the x axis). The gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). CbCas9 and CbCas9–PcrIIC1 groups are represented by Cb and Cb+P separately. Statistical analysis was calculated using two-way ANOVA with multiple comparisons; ns, P > 0.1234; *P > 0.0332; **P > 0.0021; ***P > 0.0002; ****P < 0.0001. d, In vitro cleavage of dsDNA targets with ACAAA, ACAAC and ACGTC PAM by CbCas9 and CbCas9–PcrIIC1 revealed by denaturing PAGE; aliquots were collected at time points: 0, 2, 5, 15, 30, 60, 90, and 120 min. Bottom right, bar chart showing cleavage efficiency of different dsDNA targets at 120 min. Gels shown are representative of three independent experiments with similar results and 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, In vitro cleavage of target dsDNA by CbCas9 and CTH-mutated CbCas9 with or without PcrIIC1 revealed by denaturing PAGE. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). Cb and Cb+P indicate CbCas9 and CbCas9–PcrIIC1 effectors, respectively. Cb-CTH mut and Cb-CTH mut+P indicate CTH-mutated CbCas9 and CTH-mutated CbCas9–PcrIIC1 effectors, respectively. f, Top, nucleotide sequence of CRISPR repeats. The selected GTTGT PAM is labelled with a red line. Bottom, In vitro cleavage of target dsDNA with GTTGT PAM revealed by denaturing PAGE. The gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). g, In vivo bacterial genome self-targeting assay revealed by bacterial culture plate with a series of tenfold dilution gradients. CbCas9 and CbCas9–PcrIIC1 groups with non-target spacer and target spacers are represented by Cb and Cb+P separately (Targeting PAM is shown above the plate). Data are representative of three independent experiments with similar results (n = 3 biologically independent replicates). For image and gel source data, see Supplementary Fig. 1.
