More than 50 protein families have been identified that inhibit CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-mediated adaptive immune systems. Here, we analyze the available anti-CRISPR (Acr) structures and describe common themes and unique mechanisms of stoichiometric and enzymatic suppressors of CRISPR-Cas. Stoichiometric inhibitors often function as molecular decoys of protein-binding partners or nucleic acid targets, while enzymatic suppressors covalently modify Cas ribonucleoprotein complexes or degrade immune signaling molecules. We review mechanistic insights that have been revealed by structures of Acrs, discuss some of the trade-offs associated with each of these strategies, and highlight how Acrs are regulated and deployed in the race to overcome adaptive immunity. Expected final online publication date for the Annual Review of Microbiology, Volume 74 is September 8, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional "partner" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1 NTD ) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1 NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an "anti-anti-CRISPR." In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

Highlights d Listeria anti-CRISPR protein AcrIIA1 serves as an anti-CRISPR and a vital autorepressor d The strong early acr promoter must be repressed for maximal phage fitness d AcrIIA1 allows prophages to tune Acr expression to Cas9 levels d AcrIIA1 homologs have been co-opted by host bacteria as ''anti-anti-CRISPRs''

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The viruses that infect bacteria, bacteriophages (or phages), possess numerous genes of unknown function. Genetic tools are required to understand their biology and enhance their efficacy as antimicrobials. Pseudomonas aeruginosa jumbo phage ΦKZ and its relatives are a broad host range phage family that assemble a proteinaceous “phage nucleus” structure during infection. Due to the phage nucleus, DNA-targeting CRISPR-Cas is ineffective against this phage and thus there are currently no reverse genetic tools for this family. Here, we develop a DNA phage genome editing technology using the RNA-targeting CRISPR-Cas13a enzyme as a selection tool, an anti-CRISPR gene (acrVIA1) as a selectable marker, and homologous recombination. Precise insertion of foreign genes, gene deletions, and the addition of chromosomal fluorescent tags into the ΦKZ genome were achieved. Deletion of phuZ, which encodes a tubulin-like protein that centers the phage nucleus during infection, led to the mispositioning of the phage nucleus but surprisingly had no impact on phage replication, despite a proposed role in capsid trafficking. A chromosomal fluorescent tag placed on gp93, a proposed “inner body” protein in the phage head revealed a protein that is injected with the phage genome, localizes with the maturing phage nucleus, and is massively synthesized around the phage nucleus late in infection. Successful editing of two other phages that resist DNA-targeting CRISPR-Cas systems [OMKO1 (ΦKZ-like) and PaMx41] demonstrates the flexibility of this method. RNA-targeting Cas13a system holds great promise for becoming a universal genetic editing tool for intractable phages. This phage genetic engineering platform enables the systematic study of phage genes of unknown function and the precise modification of phages for use in a variety of applications.

SUMMARYBacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse “anti-CRISPR” proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional “partner” Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an “anti-anti-CRISPR.” In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.