A truncated anti-CRISPR protein prevents spacer acquisition but not interference

Philippe, Cécile; Morency, Carlee; Plante, Pier-Luc; Zufferey, Edwige; Achigar, Rodrigo; Tremblay, Denise M.; Rousseau, Geneviève M.; Goulet, Adeline; Moineau, Sylvain

doi:10.1038/s41467-022-30310-x

Cited by 12 publications

(8 citation statements)

References 42 publications

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“…The strain JH-62 was grown overnight at 37°C, with agitation, in liquid LB medium supplemented with kanamycin 50 µg/ml (Kan50) to keep pREP4. The JH62 strain passed through a series of liquid medium passages without the ampicillin pressure, but with Kan50 to keep pREP4 and with an inoculation of 1% (61). After 12 passages, the culture was spread on LB + Kan50 plates and the colonies were streaked on LB + Kan50 and LB + Kan50 + Amp100 plates.…”

Section: Plasmid Constructions All Plasmids and Primers Used In This ...mentioning

confidence: 99%

HEPN-AbiV is an RNase in the antiphage system AbiV

Zhu,

Morency,

Picard

et al. 2024

Preprint

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Prokaryotes and eukaryotes possess defense systems, which can be either innate or acquired, to protect against viral infections. At the bacterial population level, abortive infection (Abi) serves as an innate immune defense mechanism against phage invasion. The AbiV antiviral system is prevalent in several bacterial genomes and exhibits diverse characteristics in terms of gene composition and evolution. Our investigation into the Lactococcus AbiV system revealed a novel two-component system, abiV1 and abiV2, both of which are essential for its function as a type III toxin-antitoxin system. The toxin component AbiV (product of abiV1) is an RNase belonging to the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) superfamily as it carries the consensus Rx4-6H motif. In vivo assays coupled with mass spectrometry showed that the lactococcal AbiV was expressed in the presence or absence of phages while in vitro experiments demonstrated that AbiV1 degraded ribosomal RNA but not mRNA. On the other hand, the antitoxin component (abiV2) was found to function as an RNA molecule that inhibited the nuclease activity of the AbiV1 toxin. The structural characterization of AbiV revealed that this RNase utilizes a large patch of positively charged area across the dimer to anchor RNA molecules. In addition, we showed that the AbiV N-terminal region (amino acids 1 to 23) is crucial for its RNase activity as a truncated AbiV lacking this segment adopted distinct conformational states incompatible with RNA binding. This study provided novel insights into the mode of action of the antiviral system AbiV.

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Section: Plasmid Constructions All Plasmids and Primers Used In This ...mentioning

confidence: 99%

HEPN-AbiV is an RNase in the antiphage system AbiV

Zhu,

Morency,

Picard

et al. 2024

Preprint

Self Cite

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“…For example, mutations can be introduced into the Cas protein or gRNA to prevent binding by ACR proteins, or alternative nucleases can be used that are not affected by ACR proteins. 199 Other Forms of Escape. Bacteria can also resist the CRISPR-Cas system by modifying their own DNA.…”

Section: ■ Bacterial Mechanisms Of Resistancementioning

confidence: 99%

“…These inhibitors could be used to prevent ACR proteins from binding to the CRISPR-Cas system and inhibiting its activity, thereby improving the efficiency of genome editing and other applications. , Another approach is the use of engineered CRISPR-Cas systems that are resistant to the inhibitory effects of ACR proteins. For example, mutations can be introduced into the Cas protein or gRNA to prevent binding by ACR proteins, or alternative nucleases can be used that are not affected by ACR proteins …”

Section: Bacterial Mechanisms Of Resistancementioning

confidence: 99%

CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance

et al. 2023

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The emergence of antibiotic-resistant bacterial strains is a source of public health concern across the globe. As the discovery of new conventional antibiotics has stalled significantly over the past decade, there is an urgency to develop novel approaches to address drug resistance in infectious diseases. The use of a CRISPR-Cas-based system for the precise elimination of targeted bacterial populations holds promise as an innovative approach for new antimicrobial agent design. The CRISPR-Cas targeting system is celebrated for its high versatility and specificity, offering an excellent opportunity to fight antibiotic resistance in pathogens by selectively inactivating genes involved in antibiotic resistance, biofilm formation, pathogenicity, virulence, or bacterial viability. The CRISPR-Cas strategy can enact antimicrobial effects by two approaches: inactivation of chromosomal genes or curing of plasmids encoding antibiotic resistance. In this Review, we provide an overview of the main CRISPR-Cas systems utilized for the creation of these antimicrobials, as well as highlighting promising studies in the field. We also offer a detailed discussion about the most commonly used mechanisms for CRISPR-Cas delivery: bacteriophages, nanoparticles, and conjugative plasmids. Lastly, we address possible mechanisms of interference that should be considered during the intelligent design of these novel approaches.

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“…AcrIF5 destabilizes the helical bundle domain of Cas8f in the Csy-dsDNA complex, preventing subsequent Cas2/3 recruitment 25 . In type II CRISPR-Cas systems, AcrIIA6 allosterically induce the dimerization of st1-Cas9 to reduce the binding affinity of Cas9 for DNA 26 , nevertheless, truncated AcrIIA6 does not block the interference activity of Cas9 but prevents acquisition of new spacers 27 . AcrIIA2, AcrIIA4, AcrIIC3, AcrIIC4, and AcrIIC5 inhibit the Cas9-crRNA-tracRNA complex from recognizing target DNA 28 , 29 .…”

Section: Introductionmentioning

confidence: 99%

Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5

Bi,

Su,

et al. 2024

Nat Commun

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Spacer acquisition step in CRISPR-Cas system involves the recognition and subsequent integration of protospacer by the Cas1-Cas2 complex in CRISPR-Cas systems. Here we report an anti-CRISPR protein, AcrVA5, and reveal the mechanisms by which it strongly inhibits protospacer integration. Our biochemical data shows that the integration by Cas1-Cas2 was abrogated in the presence of AcrVA5. AcrVA5 exhibits low binding affinity towards Cas2 and acetylates Cas2 at Lys55 on the binding interface of the Cas2 and AcrVA5 N-terminal peptide complex to inhibit the Cas2-mediated endonuclease activity. Moreover, a detailed structural comparison between our crystal structure and homolog structure shows that binding of AcrVA5 to Cas2 causes steric hindrance to the neighboring protospacer resulting in the partial disassembly of the Cas1-Cas2 and protospacer complex, as demonstrated by electrophoretic mobility shift assay. Our study focuses on this mechanism of spacer acquisition inhibition and provides insights into the biology of CRISPR-Cas systems.

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A truncated anti-CRISPR protein prevents spacer acquisition but not interference

Cited by 12 publications

References 42 publications

HEPN-AbiV is an RNase in the antiphage system AbiV

HEPN-AbiV is an RNase in the antiphage system AbiV

CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance

Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5

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