Anti-CRISPR phages cooperate to overcome CRISPR-Cas immunity

Bacteria evolve CRISPR-Cas immunity against bacteriophage (phage) by inserting phage-derived sequences into CRISPR loci on the host1, which can drive rapid phage extinction2. Some phages encode anti-CRISPR (acr) genes, which antagonize CRISPR-Cas immune systems by binding components of its machinery3-20, but it is less clear how these acr genes impact phage replication and epidemiology. Here we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage taking down the host CRISPR-Cas immune system for a second Acr-phage to successfully replicate. This requirement for cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points towards higher phage densities. Collectively these data help to understand how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics. Sample labels are as follows: Sample 1: Ancestral DMS3vir 2: Ancestral DMS3virAcrF1 3: Ancestral DMS3virAcrF4 4: DMS3vir evolved on WT 5: DMS3virAcrF1 evolved on WT 6: DMS3virAcrF4 evolved on WT 7: DMS3virAcrF1 evolved on BIM2 8: DMS3virAcrF4 evolved on BIM2 9: DMS3virAcrF1 evolved on BIM5 10: DMS3virAcrF4 evolved on BIM5