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Mode of Action (MoA)
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binds SpyCas9 to inhibit dsDNA cleavage.
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Evidence
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AcrIIA11 was identified as a potent anti-CRISPR protein through a two-step functional metagenomic selection that screened for elements protecting a plasmid with a kanamycin resistance gene from cleavage by SpyCas9 in E. coli. A contig derived from a Lachnospiraceae phage conferred the highest level of resistance and was significantly enriched after selection. Targeted mutagenesis of all five ORFs in the contig revealed that orf_3, named acrIIA11, was solely responsible for SpyCas9 inhibition; introducing a stop codon in this ORF eliminated the protective effect. When cloned alone, AcrIIA11 protected plasmids as effectively as known inhibitors such as AcrIIA4. In phage infection assays, AcrIIA11 expression (ECO_0000017) restored infectivity of Mu phage in E. coli expressing a SpyCas9–targeting crRNA, demonstrating functional inactivation of the CRISPR defense. Further support came from in vitro assays: purified AcrIIA11 inhibited SpyCas9-mediated DNA cleavage in a dose-dependent manner, with size exclusion chromatography (ECO_0000325) suggesting that the active form is a dimer. Pull-down assays (ECO_0006249) showed AcrIIA11 binds both apo- and sgRNA-bound forms of SpyCas9, with moderate preference for the latter. Electrophoretic mobility shift assays (ECO_0000096) demonstrated that unlike AcrIIA4, AcrIIA11 does not block SpyCas9–DNA binding but instead forms a supershifted ternary complex, indicating a novel mechanism of inhibition involving both SpyCas9 and dsDNA interaction.
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MoA Category
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binds and inhibits host defence system
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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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10.7554/eLife.46540
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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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-
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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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