Extended Data Fig. 1: Type III-A CRISPR–Cas immunity against the pG0400 conjugative plasmid and the ϕNM1γ6 phage increases the mutation frequency and rate of the staphylococcal host.

a, Schematic of the S. epidermidis RP62a type III-A locus showing the mutants analysed in this study. The CRISPR array shows repeats as black boxes and spacers as coloured, numbered boxes. b, Model of the type III-A CRISPR–Cas immune response. A Cas10 complex, composed of Cas10 and Csm2–Csm5 (in different shades of green), is loaded with a crRNA guide after processing of the transcript of the CRISPR array by Cas6 (not shown). The crRNA guide is used to direct the Cas10 complex to a complementary transcript produced by the invader following infection. Target recognition triggers two activities of Cas10. The Palm domain catalyses the conversion of ATP into a cyclic tetra- or hexa-adenosyl ring that serves as a second messenger that binds and activates Csm6, a non-specific RNase. Degradation of both cellular and invader transcripts by this nuclease results in the growth arrest of the host; this growth arrest is required for the clearance of plasmid and phage targets that have mismatches with the crRNA guide or that are transcribed either weakly or late in the phage lytic cycle. In addition, the HD domain of Cas10 is activated, leading to the non-specific degradation of ssDNA. This activity is believed to be concentrated on the ssDNA generated at the invader’s transcription bubble or within R-loops such as those formed during transcription elongation by the RNA polymerase. Finally, the Csm3 or Cmr4 subunit of the Cas10 complex cleaves the target transcript, turning off both enzymatic activities of Cas10. In this Article, we show that the ssDNase activity of type III-A CRISPR–Cas immunity can also lead to an increase in the frequency and rate of mutation of the host, presumably through degradation of other ssDNA regions of the host chromosome. c, Calculation of P values using a two-sided Mann–Whitney test of the data presented in Fig. 1b for the mutation frequency of wild-type S. epidermidis RP62a, without the two outlier data points in the pG0400 wild-type samples. Horizontal bars, median values; n (pG0400 wild type) = 14 biologically independent experiments; n (pG0400 mutant, none) = 16 biologically independent experiments. d, Staphylococcus aureus cells (around 10⁹) that were treated with ϕNM1γ6 phage and survived infection through the targeting of an early-expressed gene by the staphylococcal type II-A or type III-A CRISPR–Cas systems were seeded after 2 h of outgrowth on plates with or without rifampicin to calculate their mutation frequency. e, Staphylococcus aureus cells (fewer than 1,000) that were treated with ϕNM1γ6 phage and survived infection through the targeting of an early-expressed gene (gp12 or gp14) by the staphylococcal type II-A or type III-A CRISPR–Cas systems were seeded on plates with or without rifampicin to calculate their mutation frequency. f, Calculation of the mutation rate using the data presented in e. g, Staphylococcus aureus cells (fewer than 1,000) that were treated with ϕNM1γ6 phage and survived infection through the targeting of a late-expressed gene (gp43) by type III-A CRISPR–Cas immunity were seeded on plates with or without rifampicin to calculate their mutation frequency. In d, e, g, box limits, interquartile range; whiskers, minimum to maximum; centre line, median; dots, individual data points; n = 15 biologically independent experiments; P values obtained with a two-sided Mann–Whitney test. h, Calculation of the mutation rate using the data presented in g. In f, h, the bar graphs represent the mean; the error bars represent 95% confidence intervals.