AcrIIC1 is an 86-residue anti-CRISPR protein identified in a Neisseria meningitidis mobile-element context. It binds the active-site interface of the Cas9 HNH domain and inhibits DNA cleavage while preserving target-DNA binding. Inhibition extends to several tested Cas9 orthologues because the targeted HNH surface is conserved, but is not established for every Cas9. Disulfide-linked dimerization regulates activity in biochemical and gene-editing experiments; the physiological importance of this redox dependence in native Neisseria remains unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:27984730 Naturally Occurring Off-Switches for CRISPR-Cas9. | ACCEPT | Summary: AcrIIC1 was identified as a member of one of three anti-CRISPR families that specifically inhibit the Neisseria meningitidis CRISPR-Cas9 system, and shown to bind NmeCas9 directly. GO:0098672 is the correct and maximally specific process term, and the accession is the sequence used in the structural work on this mechanism, since PDB 5VGB chain B spans residues 1 to 86 of this entry and its primary citation is the follow-up mechanism paper. Reason: The characterized protein directly inhibits Cas9-mediated interference, supporting the existing process annotation. The biochemical mechanism is HNH-domain engagement, not a change to the annotation relationship. Supporting Evidence: PMID:27984730 Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. PMID:27984730 We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. |
| GO:0140721 nuclease inhibitor activity | IDA PMID:28844692 A Broad-Spectrum Inhibitor of CRISPR-Cas9. | NEW | Summary: AcrIIC1 binds the HNH nuclease domain of Cas9 at its active-site interface and abolishes target-strand cleavage while leaving guide loading and target DNA binding intact. Domain-swap chimeras between an AcrIIC1-binding and a non-binding Cas9 orthologue localise the interaction to HNH, the 1.50 Angstrom crystal structure shows the contacts at the active site, and the functional outcome is a DNA-bound but catalytically inactive Cas9. Reason: Direct HNH-domain binding and inhibition of DNA cleavage support nuclease inhibitor activity. This broad molecular function is appropriate even though it does not encode the retained DNA-binding activity of the target. Binding to the isolated HNH domain does not establish a requirement for preassembled Cas9-guide ribonucleoprotein. Supporting Evidence: PMID:28844692 These results indicated that the HNH domain is the primary site of interaction for AcrIIC1. PMID:28844692 AcrIIC1 binds to the active site interface of the HNH domain through several ionic and hydrogen-bonding interactions. PMID:28844692 These results suggested that AcrIIC1 traps Cas9 in its DNA-bound state, while inhibiting DNA cleavage. |
| GO:0005737 cytoplasm | IC PMID:27984730 Naturally Occurring Off-Switches for CRISPR-Cas9. | NEW | Summary: AcrIIC1 is translated from a prophage integrated in the Neisseria meningitidis chromosome and acts on the host's own Cas9, a cytoplasmic enzyme. Inhibition of NmeCas9 is demonstrated in cells, so the protein must act in the cytoplasm; all four deposited structures were determined in vitro, so the location is inferred rather than measured. Reason: GO:0030430 host cell cytoplasm, used for the phage-taxon inhibitors AcrF8, gp30 and AcrF3, is not appropriate here. This UniProt entry is assigned to Neisseria meningitidis itself, not to a phage, because the gene lies in a resident prophage, so under that taxon assignment the cell whose cytoplasm the protein occupies is its own and not a separate host. GO:0005737 is the correct component term for that situation, and the same reasoning was applied to the prophage-encoded AcrIIA4 in genes/LISMN/acrIIA4. IC rather than IDA because localization was inferred, not measured. Supporting Evidence: PMID:27984730 We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. PMID:27984730 Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. |
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Download this section (compressed HTML)Q: Does an AcrIIC1-trapped Cas9, which stays bound to its target but cannot cut, repress transcription at that locus in Neisseria, as the AcrF3-inhibited type I-F system was shown to do in Pseudomonas? If so, the inhibitor does not merely neutralise immunity but repurposes it.
Suggested experts: Lucas B. Harrington, Jennifer A. Doudna, Erik J. Sontheimer
Q: Is there a GO mechanism, perhaps an annotation extension on GO:0140721 naming the occluded domain, that could record single-domain occlusion and the survival of the target's other activities without creating a molecular-function class about protein architecture?
Q: Does the AcrIIC1 redox switch ever actually flip inside Neisseria meningitidis, or is the inactive disulphide dimer an artefact of the oxidising conditions in which the switch was characterised?
Suggested experts: Yanli Wang
Experiment: In Neisseria meningitidis, retarget NmeCas9 with a guide against a reporter promoter in strains with and without induced AcrIIC1, and in a cas9 deletion, then measure reporter transcript by RT-qPCR and Cas9 occupancy by ChIP. The prediction specific to this mechanism is repression with occupancy retained, in contrast to a DNA mimic such as AcrIIA4, which should abolish both. Running both inhibitors side by side makes the comparison a direct test of the mechanism assignment rather than of AcrIIC1 alone.
Hypothesis: An AcrIIC1-trapped Cas9 acts as a sequence-specific transcriptional repressor at its target locus in the native host.
Type: paired ChIP and transcript quantification with two inhibitor classes
Experiment: Lyse Neisseria meningitidis under strictly anaerobic conditions with immediate thiol trapping by N-ethylmaleimide, then resolve AcrIIC1 monomer and dimer by non-reducing SDS-PAGE and immunoblot across aerobic, microaerobic and oxidative-stress growth. A constant, near-complete monomer fraction would argue the switch does not operate natively and that the cysteines are conserved for another reason.
Hypothesis: The redox switch is physiological, so the native cytoplasmic pool of AcrIIC1 contains a measurable fraction of inactive disulphide dimer that shifts with growth conditions.
Type: redox-state trapping and non-reducing immunoblot
Experiment: Map the minimal AcrIIC1 region that retains HNH affinity by isothermal titration calorimetry against the purified NmeCas9 HNH domain, then test each fragment for inhibition of full-length NmeCas9 plasmid cleavage. Divergence between affinity for the isolated domain and inhibition of the intact enzyme would indicate a second contact outside HNH, which neither the chimera experiments nor the HNH-only crystal structure could have detected.
Hypothesis: AcrIIC1 binding to HNH is sufficient for inhibition, so an isolated HNH-binding fragment would inhibit full-length Cas9 as well as the intact protein does.
Type: fragment mapping with paired affinity and inhibition readouts
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether the AcrIIC1 redox switch operates in the native Neisseria meningitidis cytoplasm is undetermined; the switch was characterised with reference to eukaryotic cellular environments and gene-editing applications.
OPEN BIOLOGY
What is known: That a pair of conserved cysteines forms an inactive disulphide-linked dimer, and that the monomer is the active species, are established structurally and biochemically. What is unknown is whether the native intracellular redox potential ever moves across the switch point.
Significance: If the switch is physiological, AcrIIC1 is a conditional inhibitor and the prophage can withhold counter-defence under some conditions, which would be a regulatory layer distinct from the transcriptional Aca switch that governs other acr loci. If it is an artefact of the oxidising extracellular or eukaryotic environments used to study it, the native protein is simply constitutively active and the cysteines are doing something else.
What would resolve it: Measure the AcrIIC1 monomer-to-dimer ratio in Neisseria meningitidis lysates prepared under strictly anaerobic and thiol-trapping conditions, across growth states, and test whether cysteine-free variants alter prophage retention in a cas9-competent host.
Provenance (the field's own admissions):
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