Fig. 1: N. meningitidis CRISPR–Cas9 can restrict MDAΦ lysogenization and acquire functional viral memories.

a, The II-C CRISPR–Cas locus of N. meningitidis. Coloured squares, spacers; white diamonds, repeats; arrowheads, repeat-embedded crRNA promoters. b, MDAΦ genome organization. White arrows, phage open reading frames; black triangles, inverted repeats; red lines, protospacers targeted. c, CRISPR–Cas9 blocked MDAΦ lysogenization. MDAΦ was marked by a Kan-resistant cassette to track infection. Data are log-scale of colony-forming units (CFU) per millilitre (mean ± s.d.) of total colonies (grey bars) versus transductants (white bars) of three independent experiments. NS (P ≥ 0.05), *0.005 ≤ P < 0.05 and **P < 0.005; P values calculated using two-tailed Welch’s t-tests. d, Detecting new spacers at the leader end of CRISPR from transductants. 0 and +1, PCR amplicons from WT and elongated CRISPRs. Top, PCR primer design. The leader (L) that flanks CRISPR and contains essential elements for acquisition is depicted. Bottom, representative adaptation PCR gel; ‘adapted %’, adaptation efficiency measured as the percentage of total amplicons with new spacers on the basis of band intensities. Cas1–cas2 OE is driven by a lac regulatory system and integrated at the iga–trpB genomic site. e, Motif analysis for 3′ flanks of viral protospacers for lane 4 of d. f, Δcas9 modestly reduced efficiency. Δcas9 and its derivatives with genomic complementation of WT or mutant cas9 were assayed. Top, representative adaptation PCR gel; bottom, quantification of 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. H1024A, cas9 mutant with PAM-contacting residue H1024 mutated; PID swap, cas9 chimaera with PID switched. g, 3′ Flanking motif for viral spacers of lanes 2–6 of f. Shown is a representative sequence logo of three independent experiments that give similar results. NS, nonsignificant; NT, non-targeting; OE, overexpressing.