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Mode of Action (MoA)
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binds to Cas12a by mimicking the PAM and triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to inactivate the Cas12a complex (blocks DNA binding).
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Evidence
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AcrVA1 was ectopically expressed in both bacterial and human cells (ECO_0000017). In P. aeruginosa expressing MbCas12a and targeting phage JBD30, AcrVA1 restored phage growth. In human U2-OS cells, AcrVA1 coexpression with Cas12a and EGFP-targeting crRNA abolished gene editing, confirming strong inhibition across multiple Cas12a orthologs. Size exclusion chromatography (ECO_0000325) demonstrated that AcrVA1 binds directly to both LbCas12a alone and LbCas12a-crRNA complexes, indicating crRNA-independent binding. Functional inhibition was confirmed by in vitro DNA cleavage assays, where addition of AcrVA1 completely blocked Cas12a endonuclease activity. Cryo-EM analysis (ECO_0006181) revealed AcrVA1 inserts into the PAM-binding cleft of Cas12a, mimicking the DNA PAM duplex and inducing structural rearrangements that displace the REC1 domain. AcrVA1 also exhibited Cas12a-dependent RNase activity, cleaving the crRNA guide. Cleavage resulted in two crRNA fragments, and was abolished by mutating key active site residues (R41A, H42A, H45A -- ECO_0001113) in AcrVA1’s α2 helix. Alanine substitutions at interaction interfaces (e.g., D95A/S96A) impaired both binding and RNase function (ECO_0001113). The crRNA cleavage was independent of Cas12a’s own nuclease activity, confirming AcrVA1 contains an intrinsic RNase active site. These findings support AcrVA1-mediated inhibition of Cas12a via dual mechanisms: PAM mimicry and destruction of crRNA guidance.
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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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Moraxella bovoculi, Acidaminococcus sp. and Lachnospiraceae bacterium type V-A CRISPR-Cas
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Relevant publication(s)
DOI
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10.1126/science.aau5174,
10.1016/j.chom.2019.05.004
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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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6NMD_B
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Genome(s) encoding the protein
Protein Source
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MGE in Moraxella bovoculi 58069
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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