Figure 1. DUF1874 is an anti-CRISPR specific for cA₄ signalling.

(a) SSeV infection assay showing the DUF1874 gene SIRV1 gp29 can neutralise the type III-B system in S. islandicus. S. islandicus RJW007∆type I-A or RJW007∆type I-A∆csx1 mutant strains were challenged with SSeV, in the presence or absence of duf1874 (SIRV1 gp29) expressed on a replicative plasmid. Plaques were observed when csx1 was deleted, or when the resistant strain expressed duf1874 (n= 3 biological replicates) (Extended data Fig. 2d). (b) Schematic showing the recombinant M. tuberculosis type III-A CRISPR interference system established in E. coli. By swapping the native Csm6 ancillary nuclease for a Csx1 protein, the system can be converted from cA₆ to cA₄-mediated immunity. (c) Plasmid transformation assay (1 day’s growth) using a plasmid with a match to a spacer in the CRISPR array. If the plasmid was successfully targeted by the CRISPR system, fewer transformants were expected. Plasmids with or without the duf1874 gene were targeted successfully when cA₆ (Csm6) mediated antiviral signalling was active. In contrast, cells using a cA₄-based (Csx1) system only reduced transformation when the DUF1874 protein was not present, suggesting that DUF1874 was effective in neutralising cA₄-based CRISPR interference. The control strain lacked cOA-dependent ribonucleases. These results are representative from 2 biological replicates with 4 technical replicates each (n= 8). (d) Colony counts for transformants visible after 1- and 4-days growth in the presence or absence of DUF1874 and the indicated effector proteins. DUF1874 antagonises Csx1 but not Csm6-mediated immunity. Values and error bars reflect the mean ± standard deviation from 2 biological replicates with 4 technical replicates each (n= 8).