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Mode of Action (MoA)
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induces dimerisation of Cas12-crRNA, blocking dsDNA binding by the complex.
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Evidence
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AcrVA4 was identified from Moraxella bovoculi strain 58069 using a cell-free TXTL assay. The TXTL system includes plasmids that encode fluorescent proteins (GFP and RFP). Guide RNAs (gRNAs) are also included that direct Cas12a to specifically target and cleave the reporter plasmids. Genomic fragment GF59 increased fluorescent reporter expression, indicating inhibition of Cas12a-mediated DNA cleavage. Subsequent testing of individual open reading frames revealed GF59 candidate 2 as AcrVA4. In vitro assays confirmed that AcrVA4 inhibits dsDNA cleavage by both MbCas12a and LbCas12a, but not AsCas12a. Biochemical data further showed stronger inhibition of MbCas12a from strain 58069 than from strain 22581. In HEK293T genome editing reporter cells, AcrVA4 selectively blocked LbCas12a-induced editing, matching its in vitro activity. It did not affect SpyCas9, indicating target specificity.
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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 and Lachnospiraceae bacterium type V-A CRISPR-Cas
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Relevant publication(s)
DOI
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10.1126/science.aau5138
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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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6NM9_A
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Genome(s) encoding the protein
Protein Source
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MGE in Moraxella bovoculi 22581
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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