Extended Data Fig. 6: Characterization of the evolution mimic, co-existing arrays, and interference escaper strains.
From: Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition
a, The evolution mimic series of strains with CRISPR arrays of varying lengths expressed Cas9 to comparable levels. Top, anti-Cas9 western blot; bottom, GroEL loading control. b, flanking motif analysis for new viral spacers from Fig. 5c. c, For each array length, crRNA levels quantified from Fig. 5b and acquisition efficiencies from Fig. 5c are plotted. d, CrRNA abundance and acquisition efficiency are inversely correlated, fitted with a one-phase decay non-linear regression model in Prism. e, Schematics of array-coexistence strains used for MDAΦ infection-acquisition test under cas1-2 induction in f. Genotypes at endogenous CRISPR locus and the 2nd CRISPR array inserted at the NICS genomic site are depicted. f, Total acquisition efficiency was evenly distributed between co-existing arrays, with longer 2nd arrays progressively reducing acquisition at both CRISPR loci within the same cell. Data shown 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. g, Donut chart of interference escaping mechanisms. Purple, cas9 mutations (38/62); blue, CRISPR array collapse (13/62); red, target mutation or deletion (8/62); green, unclear (5/62). h, Interference escapers with mutated cas9 (e2, e3, e4) are permanently defective for interference, as revealed by interference re-test using transforming DNA bearing protospacers 6 or 25. i, Array-contracted escapers expressed Cas9 protein to comparable levels as the WT array. j, 3′ flanking motif for new viral spacers of Fig. 5i.
