Structures and Strategies of Anti-CRISPR-Mediated Immune Suppression.

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.

Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex. IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu.

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.

Silencing of toxic gene expression by Fis.

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.

Binding Affinity Quantifications of the Bacteriophage Mu DNA Modification Protein Mom Using Microscale Thermophoresis (MST).

Epigenetic modifications play diverse roles in biological systems. Nucleic acid modifications control gene expression, protein synthesis, and sensitivity to nucleic acid-cleaving enzymes. However, the mechanisms underlying the biosynthesis of nucleic acid modifications can be challenging to identify. Studying protein-ligand interactions helps decipher biosynthetic and regulatory pathways underlying biological reactions. Here, we describe a fluorescence labeling-based quantitative method for unraveling the biomolecular interactions of bacteriophage Mu DNA modification protein Mom with its ligands, using microscale thermophoresis (MST). Compared to traditional methods for studying protein-biomolecular interactions, MST requires significantly lower sample amounts, volumes, and analysis time, thus allowing screening of a large number of candidates for interaction with a protein of interest. Another distinguishing feature of the method is that it obviates the need for protein purification, often a time- and resource-consuming step, and works well with whole or partially purified cell extracts. Importantly, the method is sensitive over a broad range of molecular affinities while offering great specificity and can be used to interrogate ligands ranging from metal ions to macromolecules. Although we established this method for a DNA modification protein, it can easily be adapted to study a variety of molecular interactions engaged by proteins.

Bacteriophage genome engineering with CRISPR-Cas13a.

Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.

Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor.

Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1NTD is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1NTD is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a "Cas9 sensor," tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1NTD homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as "anti-anti-CRISPRs," repressing phage anti-CRISPR deployment.

Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.