The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages1–3. We identified the first examples of proteins produced by phages that inhibit a CRISPR–Cas system4. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR–Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR–Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase–nuclease and preventing its recruitment to the DNA-bound CRISPR–Cas complex. In vivo, this anti-CRISPR can convert the CRISPR–Cas system into a transcriptional repressor, providing the first example—to our knowledge—of modulation of CRISPR–Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR–Cas function.

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-mediated adaptive immune systems for protection from viral infection and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here we report the cryo-electron microscopy structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

Significance Prokaryotes have adaptive immune systems that rely on CRISPRs (clustered regularly interspaced short palindromic repeats) and diverse CRISPR-associated ( cas ) genes. Cas1 and Cas2 are conserved components of CRISPR systems that are essential for integrating fragments of foreign DNA into CRISPR loci. In type I-F immune systems, the Cas2 adaptation protein is fused to the Cas3 interference protein. Here we show that the Cas2/3 fusion protein from Pseudomonas aeruginosa stably associates with the Cas1 adaptation protein, forming a 375-kDa propeller-shaped Cas1–2/3 complex. We show that Cas1, in addition to being an essential adaptation protein, also functions as a repressor of Cas2/3 nuclease activity and that foreign DNA binding by the CRISPR RNA-guided surveillance complex activates the Cas2/3 nuclease.

The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide.