Extended Data Fig. 4: R26 allele is crRNA null.
From: Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition
a, Different crRNA biogenesis pathways in types II-A and II-C systems. Left, II-A crRNA is transcribed from an external promoter within the leader, producing crRNAs and a leader-derived extraneous crRNA. Right, II-C crRNA is transcribed from repeat-embedded internal promoters and initiates from the 6th nt of each spacer. b, II-C R26 allele is predicted to not produce any mature crRNA or crRNA mimic. c, northern blot validating b. Total RNAs of the R26 strain were probed for crRNA, tracrRNA, and 5S loading control. ΔCRISPR, Δtracr, and Δcas9 strains are controls. d, Schematic of dcas9-encoding trains used in panels e-f. e, R26 triggered super-adaptation is independent of interference or Cas9’s nuclease activity. Strains isogenic to those in Fig. 2e but encode WT cas9 were assayed for MDAΦ infection-acquisition, with or without IPTG induction of cas1-2. Data are shown as in Fig. 2e. 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. f, 3′ flanking motif for new spacers from panel e. g-h, Super-adaptation in Fig. 2e is not due to changes in Cas9 or Cas1 protein levels. Anti-Cas1 (g, +/- IPTG induction) and anti-Cas9 (h) western blots showed comparable protein levels in FL CRISPR vs. R26 strains. GroEL is loading control.
