Extended Data Fig. 10: ApoCas9 and tracrRNA-crRNA play conserved roles in acquisition across divergent II-C systems.

a, Schematic of strains used. b, Phylogeny of II-C cas9 orthologs tested. Percentages of protein sequence identity relative to NmeCas9 are in parentheses. II-A SpyCas9 is included as an outgroup. c, Top, anti-Flag western blots confirmed the expression of II-C cas9 orthologs in Neisseria, and that their levels were not affected by the respective sgRNA. Asterisk, expression of CjeCas9, unlike other orthologs, required aTc induction of pTet to become detectable by western blot. GroEL, loading control. d, ApoCjeCas9 failed to induce super-adaptation. Left, a representative adaptation PCR. Right, quantification of adaptation efficiencies. Data are mean ± s.d., n = 3. NS, not significant (P ≥ 0.05), * 0.005 ≤ P < 0.05, ** P < 0.005; P values calculated by two-tailed Welch’s t-tests. e, 3′ flanking motifs analysis of new viral spacers from panel d. f, Multiple II-C Cas9s stimulated super-adaptation, which was repressed to baseline level by their respective sgRNAs. Top, representative adaptation PCR. Bottom, quantification of adaptation efficiencies, mean ± s.d., n = 3. NS, not significant (P ≥ 0.05), * 0.005 ≤ P < 0.05, ** P < 0.005; P values calculated by two-tailed Welch’s t-tests. g, 3′ flanking motifs for new viral spacers from panel f. h, CRISPR interference assay showed that WT Cas9 and sgRNA- expressing strains are competent for interference and requires the corresponding PAMs. Data are shown as in Extended Data Fig. 2k.