Biology subjects

Bondy-Denomy, J.

Publications and source records attributed to Bondy-Denomy, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile

CRISPR-Cas systems provide their prokaryotic hosts with adaptive immunity against mobile genetic elements. Many bacteriophages encode anti-CRISPR (Acr) proteins that inhibit host defense. The identification of Acr proteins is challenging due to their small size and high sequence diversity, and only a limited number has been characterized to date. In this study, we report the discovery of a novel Acr protein, AcrIB2, encoded by the {varphi}CD38-2 Clostridioides difficile phage that efficiently inhibits interference by the type I-B CRISPR-Cas system of the host and likely acts as a DNA mimic. Most C. difficile strains contain two cas operons, one encoding a full set of interference and adaptation proteins and another encoding interference proteins only. Unexpectedly, we show that only the partial operon is required for interference and is subject to inhibition by AcrIB2.

microbiology↗

bioRxiv2023

Parallelized screening of virus accessory genes reveals diverse defense and counter-defense mechanisms

Viruses have been evolving host-modifying factors for billions of years. Genomes of bacterial and archaeal viruses are replete with fast-evolving, uncharacterized accessory genes (AGs), most of which likely antagonize host defenses or other viruses1, 2. Systematic investigation of AGs could uncover a multitude of biological mechanisms involved in virus-host competition, but AG identification in genomic databases remains a challenge. We developed an integrated computational and high-throughput discovery platform to identify AGs in virus genomes and assay their functions in complementary phage infection-dependent and -independent contexts. Our approach showcases how phages interact with the principal layers of antiviral immunity, including cell surface modifications, restriction systems, and abortive infection (Abi) mechanisms, which operate simultaneously in the same host. We discovered multiple Enterobacteriophage AGs associated with counter-defense functions that activate rather than inhibit antiviral immunity in cells, including the surprising finding that anti-restriction AGs elicit programmed cell death (PCD) activity of some restriction-modification (R-M) systems. We propose that counter-defense AGs that trigger PCD create a conundrum for phages whereby keeping the AGs causes PCD but losing them exposes the phage to restriction by bacteria. Strategies employed by viruses to avoid this double jeopardy could be an important factor in virus evolution that remains to be explored.

microbiology↗

bioRxiv2023

Anti-CRISPR protein mediated degradation of Cas9 in human cells

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. Optimized expression of AcrIIA1 in human cells provided robust inhibition of SpyCas9 editing but had no impact on Type V CRIPSR-Cas12a, consistent with its binding site on the HNH domain. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

biochemistry↗

bioRxiv2023

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages ({phi}KZ and{phi} PA3) in Pseudomonas aeruginosa. This enabled the detection >6000 unique host-pathogen and >200 pathogen-pathogen interactions for each phage, encompassing >50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a{phi} PA3 complex showing strong structural and sequence similarity to{phi} KZ nvRNAp, suggesting{phi} PA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the virion packaged and injected proteome by identifying 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for interactions between KZ-like phage proteins and the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction (https://phagemap.ucsf.edu/). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of hostpathogen interactomes and protein complex dynamics upon infection.

systems biology↗

bioRxiv2022

Translation-dependent downregulation of Cas12a mRNA by an anti-CRISPR protein

Bacteria have evolved multiple defense systems, including CRISPR-Cas, to cleave the DNA of phage and mobile genetic elements (MGE). In turn, phage have evolved anti-CRISPR (Acr) proteins that use novel and co-opted mechanisms to block DNA binding or cleavage. Here, we report that an anti-CRISPR (AcrVA2) unexpectedly inhibits Cas12a biogenesis by triggering translation-dependent destruction of its mRNA. AcrVA2 specifically clears the mRNA of Cas12a by recognizing and binding its N-terminal polypeptide. Mutating conserved N-terminal amino acids in Cas12a prevents binding and inhibition by AcrVA2 but also decreases Cas12a anti-phage activity. This mechanism therefore enables AcrVA2 to specifically inhibit divergent Cas12a orthologs while constraining its ability to escape inhibition. AcrVA2 homologs are found on diverse MGEs across numerous bacterial classes, typically in the absence of Cas12a, suggesting that this protein family may induce similar molecular outcomes against other targets. These findings reveal a new gene regulatory strategy in bacteria and create opportunities for polypeptide-specific gene regulation in prokaryotes and beyond.

microbiology↗

bioRxiv2022

A family of novel immune systems targets early infection of nucleus-forming jumbo phages

Jumbo bacteriophages of the [ovbar]KZ-like family are characterized by large genomes (>200 kb) and the remarkable ability to assemble a proteinaceous nucleus-like structure. The nucleus protects the phage genome from canonical DNA-targeting immune systems, such as CRISPR-Cas and restriction-modification. We hypothesized that the failure of common bacterial defenses creates selective pressure for immune systems that target the unique jumbo phage biology. Here, we identify the "jumbo phage killer" (Juk) immune system that is deployed by a clinical isolate of Pseudomonas aeruginosa to resist [ovbar]KZ. Juk immunity rescues the cell by preventing early phage transcription, DNA replication, and nucleus assembly. Phage infection is first sensed by JukA (formerly YaaW), which localizes rapidly to the site of phage infection at the cell pole, triggered by ejected phage factors. The effector protein JukB is recruited by JukA, which is required to enable immunity and the subsequent degradation of the phage DNA. JukA homologs are found in several bacterial phyla and are associated with numerous other putative effectors, many of which provided specific anti-[ovbar]KZ activity when expressed in P. aeruginosa. Together, these data reveal a novel strategy for immunity whereby immune factors are recruited to the site of phage protein and DNA ejection to prevent phage progression and save the cell.

microbiology↗

bioRxiv2022

The {varphi}PA3 Phage Nucleus is Enclosed by a Self-Assembling, 2D Crystalline Lattice

A growing number of jumbo bacteriophages, with genomes exceeding 200 kb, have been found to establish a Phage Nucleus--a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the Phage Nucleus while nucleotide synthesis, translation, and numerous other host and exogenous proteins are effectively excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that fragments of the Phage Nucleus isolated from {phi}PA3 infected Pseudomonas aeruginosa cells form a square lattice and demonstrate that the recombinantly purified primary Phage Nuclear Enclosure (PhuN) protein spontaneously assembles into sheets also constructed from a square lattice which we resolve to 3.8 [A] by cryo-EM. Our structure reveals that the flexible termini and large loops mediate adaptable inter-tetramer contacts that drive shell assembly into a C2-symmetric lattice. While the interfaces between subunits are mostly well packed, two of the interfaces are open, forming clear channels that likely have important functional implications for the transport of proteins, mRNA, and small molecules.

biochemistry↗

bioRxiv2022

Bacteriophages antagonize cGAS-like immunity in bacteria

The recently discovered cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is related to eukaryotic cGAS-STING anti-viral immunity and is present in diverse prokaryotes. However, our understanding of how CBASS detects, inhibits, and co-evolves with phages is limited because CBASS function has only been studied in reconstituted heterologous systems. Here, we identify a phage-encoded CBASS antagonist (acbIIA1, anti-cbass type II-A gene 1) necessary for phage replication in the presence of endogenous CBASS immunity in Pseudomonas aeruginosa. acbIIA1 homologs are encoded by numerous lytic and temperate phages infecting Gram-negative bacteria. Deletion of acbIIA1 renders multiple phages susceptible to CBASS, but phages can then escape immune function via mutations in the major capsid gene. These mutants suggest that CBASS is activated by, or targets, the late-expressed phage capsid. Together, we establish a native model system to study CBASS and identify a common phage-encoded CBASS antagonist, demonstrating that CBASS is a bona fide anti-phage immune system in nature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/486325v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a4bb64org.highwire.dtl.DTLVardef@10ade0forg.highwire.dtl.DTLVardef@78e1b0org.highwire.dtl.DTLVardef@28f556_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗

bioRxiv2022

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities are in vitro irregularities.

microbiology↗

bioRxiv2022

RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering

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 {Phi}KZ 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 {Phi}KZ 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 ({Phi}KZ-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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/480438v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b1b4a6org.highwire.dtl.DTLVardef@cb0e08org.highwire.dtl.DTLVardef@1064217org.highwire.dtl.DTLVardef@1dcfbb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements, like bacteriophages and plasmids. To overcome CRISPR immunity, phages have evolved diverse families of anti-CRISPR proteins (Acrs), each of which inhibits the nucleic acid binding or cleavage activities of specific Cas protein families. Recently, Lin et al. (2020) described the discovery and characterization of 7 different Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems, which use the nuclease Cas13a to perform RNA-guided RNA cleavage. In this Matters Arising article, we detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses, as well as reported experiments involving bacterial strains that are impossible to construct. The authors were unable to provide their published strains with which we might reproduce their experiments. We independently tested the Acr sequences described in Lin et al. (2020) in two different Cas13 inhibition assays, but could not detect anti-CRISPR activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are bona fide type VI anti-CRISPR proteins.

molecular biology↗

bioRxiv2021

F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

microbiology↗

bioRxiv2020

Direct observation of genome surveillance by CRISPR-Cas in bacteria

CRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, 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.

microbiology↗

bioRxiv2020

Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa

Bacteria deploy multiple defense mechanisms to prevent the invasion of mobile genetic elements (MGEs). CRISPR-Cas systems use RNA-guided nucleases to target MGEs, which in turn produce anti-CRISPR (Acr) proteins that inactivate Cas protein effectors. The minimal component Type I-C CRISPR-Cas subtype is highly prevalent in bacteria, and yet a lack of a tractable in vivo model system has slowed its study, the identification of cognate Acr proteins, and thus our understanding of its true role in nature. Here, we describe MGE-MGE conflict between a mobile Pseudomonas aeruginosa Type I-C CRISPR-Cas system always encoded on pKLC102-like conjugative elements, which are large mobile islands, and seven new Type I-C anti-CRISPRs (AcrIF2*, AcrIC3-IC8) encoded by phages, other mobile islands, and transposons. The P. aeruginosa Type I-C system possesses a total of 300 non-redundant spacers (from 980 spacers total) across the 42 genomes analyzed, predominantly targeting P. aeruginosa phages. Of the seven new Type I-C anti-CRISPRs, all but one are highly acidic, and four have surprisingly broad inhibition activity, blocking multiple distantly related P. aeruginosa Type I CRISPR system subtypes (e.g. I-C and I-F, or I-C and I-E), including AcrIF2 (now, AcrIF2*), a previously described DNA mimic. Anti-type I-C activity of AcrIF2* was far more sensitive to mutagenesis of acidic residues in AcrIF2* than anti-type I-F activity, suggesting distinct binding mechanisms for this highly negatively charged protein. Five of the seven Acr proteins block DNA-binding, while the other two act downstream of DNA-binding, likely by preventing Cas3 recruitment or activity. For one such Cas3 inhibitor (AcrIC3), we identify a novel anti-CRISPR evasion strategy: a cas3-cas8 gene fusion, which also occurs in nature. Collectively, the Type I-C CRISPR spacer diversity and corresponding anti-CRISPR response, all occurring on Pseudomonas MGEs, demonstrates an active co-evolutionary battle between parasitic elements.

microbiology↗

bioRxiv2020

Discovery of multiple anti-CRISPRs uncovers anti-defense gene clustering in mobile genetic elements

Many prokaryotes employ CRISPR-Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have evolved Anti-CRISPR (Acr) proteins to bypass this immunity, yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we uncover 11 new type I anti-CRISPR genes encoded on numerous chromosomal and extrachromosomal mobile genetic elements within Enterobacteriaceae and Pseudomonas. Candidate genes were identified adjacent to anti-CRISPR associated gene 5 (aca5) and assayed against a panel of six type I systems: I-F (Pseudomonas, Pectobacterium, and Serratia), I-E (Pseudomonas and Serratia), and I-C (Pseudomonas), revealing the type I-F and/or I-E acr genes and a new aca (aca9). We find that acr genes not only associate with other acr genes, but also with inhibitors of distinct bacterial defense systems. These genomic regions appear to be "anti-defense islands", reminiscent of the clustered arrangement of "defense islands" in prokaryotic genomes. Our findings expand on the diversity of CRISPR-Cas inhibitors and reveal the potential exploitation of acr loci neighborhoods for identifying new anti-defense systems.

microbiology↗

bioRxiv2020

Vast diversity of anti-CRISPR proteins predicted with a machine-learning approach

Bacteria and archaea evolve under constant pressure from numerous, diverse viruses and thus have evolved multiple defense systems. The CRISPR-Cas are adaptive immunity systems that have been harnessed for the development of the new generation of genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including numerous, diverse anti-CRISPR proteins (Acrs) that can inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small, highly variable proteins which makes their prediction a formidable task. We developed a machine learning approach for comprehensive Acr prediction. The model showed high predictive power when tested against an unseen test set that included several families of recently discovered Acrs and was employed to predict 2,500 novel candidate Acr families. An examination of the top candidates confirms that they possess typical Acr features. One of the top candidates was independently tested and found to possess anti-CRISPR activity (AcrIIA12). We provide a web resource (http://acrcatalog.pythonanywhere.com/) to access the predicted Acrs sequences and annotation. The results of this analysis expand the repertoire of predicted Acrs almost by two orders of magnitude and provide a rich resource for experimental Acr discovery.

microbiology↗

bioRxiv2019

A minimal CRISPR-Cas3 system for genome engineering

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an "all-in-one" vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an "anti-anti-CRISPR" strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

molecular biology↗

bioRxiv2019

Listeria phages induce Cas9 degradation to protect lysogenic genomes

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 (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.

microbiology↗