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Mode of Action (MoA)
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modifies MGE DNA topology.
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Evidence
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AcrIIA22 was identified as a novel anti-CRISPR through a functional metagenomic screen using a two-plasmid system in E. coli, where its expression allowed retention of a kanamycin-resistant target plasmid otherwise eliminated by SpyCas9. Deletion (ECO_0001038) or mutation of acrIIA22 (orf_1) abolished this protective effect, confirming its necessity and sufficiency for CRISPR inhibition. AcrIIA22 did not bind or inhibit SpyCas9 in vitro directly, suggesting an alternative mechanism. Structural analysis via X-ray crystallography (PDB: 7JTA) revealed homology to PC4-like nucleic acid–binding proteins, predicting a role in DNA interaction. Biochemical assays demonstrated that AcrIIA22 functions as a DNA nickase, converting supercoiled plasmids into relaxed forms in vivo and in vitro. Mutations that impaired this nicking activity (e.g., D14A or natural variant AcrIIA22a) also reduced anti-CRISPR activity in bacterial plasmid protection assays, confirming that DNA nicking underlies its function. Pre-nicked plasmids became resistant to SpyCas9 cleavage in vitro, linking AcrIIA22’s activity to altered DNA topology and impaired Cas9 R-loop formation.
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MoA Category
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modifies phage molecules to avoid recognition
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Subtype(s) of the defence system(s) inhibited by the protein
Defence Subtype
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Streptococcus pyogenes type II-A CRISPR-Cas
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Relevant publication(s)
DOI
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10.1371/journal.pbio.3001428
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Other components of the anti-defence system
Multicomponent System
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-
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Known structure in PDB
PDB ID
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7JTA_A
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Genome(s) encoding the protein
Protein Source
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Metagenome
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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