Fig. 5: ApoCas9 assists CRISPR array neogenesis and replenishment of collapsed arrays.
From: Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition
a, Schematic of serial arrays of varying lengths to imitate CRISPR evolution from a single lone repeat. The WT array in N. meningitidis strain 8013 has 25 spacers. b, Northern blot showing the trend of mature crRNA increase. Total RNAs extracted were probed for crRNA (top), tracrRNA (bottom) and 5S rRNA loading control (middle). The dashed grey line indicates non-adjacent lanes from the same gel. c, The efficiency of spacer acquisition inversely correlates with CRISPR array length. Top, a representative adaptation PCR gel; bottom, quantification 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 using two-tailed Welch’s t-tests. d, Schematic for CRISPR interference of a beneficial horizontally transferred trait. The transforming DNA bears a chloramphenicol (Cm) resistance marker (green) and protospacer (ps) 6 (orange), together flanked by homology arms (black) for recombination into the host genome. e, Transformation interference assay using high amounts of DNA (3 μg) to obtain escapers. Y axis, log-scale of CFU per millilitre (mean ± s.d.; n = 3) for total cells (blue bars) and transformants (red bars). f, PCR diagnostic of CRISPR length in representative escapers (e). Array truncations were confirmed by Sanger sequencing and illustrated at the bottom. Red, spacer 6. g, Interference assay retesting escapers using different targets, protospacers 25 (left) versus 6 (right). Data plotted as in e. h, CRISPR array-collapsed escapers produced much less crRNA than WT strain. Northern blot was conducted and shown as in b. i, The array-collapsed escapers, assayed in a Cas1–2 overexpression context, elevated their spacer acquisition efficiencies. Data are shown as in c.
