Fig. 3: The crRNA–tracrRNA pair controls the adaptation-stimulatory activity of apoCas9.
From: Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition
a, Schematic of the strains used. Blue box, cas9 complemented at the iga–trpB genomic locus, alone or with RNA partners. Cas9 was driven by leaky expression from the Tet promoter, and tracr and sgRNA were driven from a copy of the tracrRNA promoter. b, Northern blot confirming the absence or presence of small RNAs in accordance with strain genotypes. The orange stars denote sgRNA, detectable by both anti-repeat (top) and anti-tracr (middle) northern probes. 5S ribosomal RNA (rRNA), loading control (bottom). c, ApoNmeCas9 was the driver of super-adaptation and was suppressed by the crRNA–tracrRNA pair. Top, a representative adaptation PCR gel. Bottom, bar graph of adaptation efficiencies. Data are mean ± s.d., n = 3. NS (P ≥ 0.05), *0.005 ≤ P < 0.05 and **P < 0.005; P values calculated by two-tailed Welch’s t-tests. d, 3′ Flanking motif analysis for new viral spacers from c, showing successful PAM enrichment for super-adaptation and baseline adaptation conditions.
