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Mode of Action (MoA)
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interacts with the WED domain of SpyCas9, where it spatially obstructs conformational changes of the WED and PI domains, thereby inhibiting SpyCas9 from recognising the protospacer adjacent motif (PAM) and unwinding double-stranded DNA.
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Evidence
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AcrIIA32 was cloned from a Streptococcus phage-associated mobile element and tested for its ability to inhibit SpyCas9 and St3Cas9. In plasmid interference assays, E. coli expressing either Cas9 ortholog along with targeting sgRNA displayed growth inhibition due to plasmid cleavage. Co-expression of AcrIIA32 restored growth, indicating effective inhibition. In vitro cleavage assays confirmed that AcrIIA32 inhibited both DNA binding and cleavage by Cas9. EMSAs showed no Cas9–DNA complex formation in the presence of AcrIIA32. Cryo-electron microscopy structure supports direct binding (ECO_0006181).
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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 thermophilus and Streptococcus pyogenes type II-A CRISPR-Cas
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Relevant publication(s)
DOI
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10.1093/nar/gkac099,
10.1007/s11427-024-2607-8
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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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8YE6_B
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Genome(s) encoding the protein
Protein Source
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MGE in Streptococcus uberis NZ01
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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