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Mode of Action (MoA)
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binds to Cas9, blocking DNA binding
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Evidence
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AcrIIC5Smu was identified as a highly potent NmeCas9 inhibitor through in vitro cleavage assays where it blocked DNA cleavage at lower concentrations than AcrIIC4Hpa, achieving full inhibition at ~7-fold molar excess. In phage immunity assays, AcrIIC5Smu expression fully restored infectivity of phage Mu in E. coli strains expressing NmeCas9 and HpaCas9, indicating strong cross-species inhibition. Although no direct interaction with Cas9 was observed in bacterial co-purification, co-immunoprecipitation (ECO_0005644) from mammalian cell lysates confirmed physical binding between AcrIIC5Smu and NmeCas9. In HEK293T cells, AcrIIC5Smu expression abolished genome editing by NmeCas9 at multiple loci, while editing by SpyCas9 remained unaffected, demonstrating high specificity. Biochemical assays showed that AcrIIC5Smu does not disrupt sgRNA loading but prevents target DNA binding, reducing NmeCas9 DNA affinity by ~6-fold. In live-cell imaging, AcrIIC5Smu eliminated telomeric focus formation by dNmeCas9 while sparing dSpyCas9, verifying functional inhibition of DNA binding in human cells and supporting its utility as a precise anti-CRISPR off-switch.
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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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Neisseria meningitidis type II-C CRISPR-Cas
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Relevant publication(s)
DOI
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10.1128/mBio.02321-18
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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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8JB9
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Genome(s) encoding the protein
Protein Source
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MGE in Simonsiella muelleri
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Defence system(s) inhibited by the protein
Defences
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CRISPR-Cas
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