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Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains., Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector’s toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr’s pivotal role in immunity., Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR–Cas, restriction–modification and a growing list of mechanistically diverse systems, which constitute the bacterial ‘immune system’. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond., To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR–Cas, restriction–modification and a growing list of mechanistically diverse systems, which constitute the bacterial ‘immune system’. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond.

Many bacteria use CRISPR–Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR–Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR–Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR–Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR–Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6., Many bacteria use CRISPR–Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR–Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR–Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR–Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR–Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6.

Many bacteria use CRISPR–Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR–Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR–Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR–Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR–Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6., Many bacteria use CRISPR–Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR–Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR–Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR–Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR–Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6.