AcrIIA5 is a 140-residue anti-CRISPR protein encoded by the virulent Streptococcus thermophilus phage D4276. It adopts a novel seven-stranded beta-sheet and two-helix fold preceded by a basic, intrinsically disordered N-terminal region of about 22 residues. Inside the infected bacterial cell it binds the guide-RNA-loaded Cas9 ribonucleoprotein and blocks cleavage of the target DNA duplex, while leaving guide loading and target-DNA engagement intact; it does not degrade the guide RNA itself and does not act on apo-Cas9. The disordered tail, which supplies both the length needed for association and the positive charges needed for inhibition, and a mobile beta3-beta4 loop in the folded core are the functional determinants. Inhibition extends across type II-A, II-B and II-C Cas9 orthologues, making it one of the broadest-spectrum Cas9 inhibitors known, and it operates in its native host, in heterologous bacteria and in human cells. The biological consequence is escape of the phage genome from host CRISPR-Cas9 immunity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003723 RNA binding | IPI PMID:32544231 Intrinsic disorder is essential for Cas9 inhibition of anti-... | MODIFY | Summary: The DisProt IPI row records an interaction between AcrIIA5 and a Cas9 single-guide RNA. In the cited paper the measured interaction is with the preformed Cas9-sgRNA ribonucleoprotein, detected as an sgRNA super-shift in an electrophoretic mobility shift assay (Figure 4A) and corroborated by amide line-broadening on NMR titration (Figure 4E). The same paper shows that AcrIIA5 has no ribonuclease activity on free sgRNA and does not bind apo-Cas9, so the RNA is engaged only in the context of the holo-enzyme. The partner is a ribonucleoprotein complex, not a free RNA ligand. Reason: The essence of the annotation is sound - there is a direct, experimentally measured physical interaction with a guide-RNA-containing entity - but GO:0003723 asserts binding to RNA as such, which this paper does not establish and in part argues against, since AcrIIA5 binds only the assembled complex. GO:0043021 ribonucleoprotein complex binding names the entity actually assayed and is the term this repository already uses for the equivalent AcrIIA4-Cas9/sgRNA relationship in genes/LISMN/acrIIA4. This is a lateral correction of the binding partner's identity rather than a refinement, so the replacement is not a descendant of the original term. Proposed replacements: ribonucleoprotein complex binding Supporting Evidence: PMID:32544231 Titration of AcrIIA5 into Cas9βsgRNA resulted in sgRNA super-shifts owing to the ternary complex formation, demonstrating a direct interaction between AcrIIA5 and Cas9βsgRNA PMID:32544231 We note that AcrIIA5 interacted only with the preformed Cas9βsgRNA complex, and not with apo-Cas9. PMID:32544231 Treatment of free sgRNA or the Cas9βsgRNA complex with AcrIIA5 did not digest sgRNA, and the AcrIIA5-treated sgRNA bands remained intact in the urea gel |
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:28785032 An anti-CRISPR from a virulent streptococcal phage inhibits ... | ACCEPT | Summary: CACAO row, evidence ECO:0007040 (plaque assay evidence used in manual assertion). What the cited paper shows for this gene: phage D4276 escaped CRISPR immunity in Streptococcus thermophilus more than 40,000-fold more efficiently than related phages, systematic cloning of its ORFs identified a single gene wholly responsible for that escape, and the cloned product also abolished immunity conferred by the heterologous Streptococcus pyogenes Cas9 in another genus. That gene product is the 140-residue protein of this entry (D4276_028). The CACAO term matches the assay exactly: GO:0098672 is the most specific GO term for a symbiont protein neutralising host CRISPR-Cas immunity, and plaquing on CRISPR-immunised versus naive hosts measures that process directly rather than a downstream phenotype. The chosen ECO code is also an unusually precise one for the assay actually performed. Reason: The claim is correct, maximally specific, and directly supported by the cited experiments on this protein, so this is neither a phenotype-read-as-process nor a wrong-granularity case. The only defect is the GAF qualifier. acts_upstream_of_or_within_positive_effect describes a gene product that influences a process from outside it, whereas AcrIIA5 itself carries out the suppression, which is what the two involved_in rows for the same term correctly say. That is a source-record field error, recorded in source_defects, and not a reason to reject the claim. Supporting Evidence: PMID:28785032 we found one that evaded the CRISPR-encoded immunity >40,000Γ more often than the others PMID:28785032 Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. PMID:28785032 This Acr completely abolishes SpCas9-mediated immunity in our assays. |
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:28785032 An anti-CRISPR from a virulent streptococcal phage inhibits ... | ACCEPT | Summary: CACAO row, evidence ECO:0007040 (plaque assay), same paper and term as the row above but with the correct involved_in qualifier. What the paper shows for this gene is the discovery experiment: an Acr from virulent phage D4276 that is solely responsible for abolishing both native Streptococcus thermophilus CR1 immunity and heterologous SpCas9 immunity in phage challenge assays. The CACAO term matches that result at the right granularity. The cached publication is abstract-only, so the figure numbers behind the plaquing data cannot be cited, but the abstract states the result for this protein outright and the student curator will have read the full text. Reason: Correct, maximally specific term with direct experimental support on this protein. Rows sharing this term are all accepted; this is the canonical representation of the gene's process and it is retained as a core function. Supporting Evidence: PMID:28785032 we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus PMID:28785032 We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. |
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IDA PMID:30046034 Widespread anti-CRISPR proteins in virulent bacteriophages i... | ACCEPT | Summary: CACAO row, evidence ECO:0000314 (direct assay). The cited paper is titled and framed around AcrIIA6, a different anti-CRISPR family, so this row initially looks like a paper-not-about-the-gene case. It is not. AcrIIA5_D4276 (140 aa, this exact protein) is assayed side by side with the AcrIIA6 proteins throughout, appears as its own row in the Table 1 activity summary, and is the subject of specific conclusions. Plaquing assays on Streptococcus thermophilus immunised through CR1 or CR3 and on Lactococcus lactis carrying plasmid-borne SpCas9 (Figure 4) show full restoration of phage titre, and human K562 cell editing assays (Figure 5) show inhibition of SpCas9 and St1Cas9. The CACAO term matches the assays at the correct granularity. Reason: Independent direct-assay support for the same claim in a second publication, in the native host genus, in a heterologous bacterium and in human cells. One important nuance for this orthologue, which the term does not need to capture but the review should record. D4276 AcrIIA5 is potent against St1Cas9 and SpCas9 but only weakly active against St3Cas9, where the 87% identical D1126 orthologue is fully active. That is a quantitative limit on breadth, not a reason to qualify the process term. Supporting Evidence: PMID:30046034 All AcrIIA6 and AcrIIA5 orthologs completely restored the plaquing efficiency of phages targeted by the CR1 system of S. thermophilus (Fig. 4), indicating they are blocking the activity of St1Cas9. PMID:30046034 Both AcrIIA5s almost completely abolished SpCas9 activity in this bacterial system PMID:30046034 Notably, AcrIIA5D4276 comes from a phage infecting a S. thermophilus host which lacks the CR3 locus, and its activity is limited to a 10-fold inhibition of CR3 activity25. PMID:30046034 we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells |
| GO:0140678 molecular function inhibitor activity | IMP PMID:32544231 Intrinsic disorder is essential for Cas9 inhibition of anti-... | MODIFY | Summary: The DisProt row captures the right molecular function in kind but stops at the top of the inhibitor-activity branch. The cited paper establishes both what is inhibited and by what. Purified AcrIIA5 alone completely blocks Cas9-sgRNA cleavage of a double-stranded DNA target in vitro (Figure 3A), and a graded series of N-terminal truncations and charge-neutralising mutations of the disordered region loses that inhibition (Figures 3C, 3E, 3F), which is the mutant-phenotype reasoning behind the IMP code. Two further papers establish the inhibited activity as DNA cleavage specifically, with target-DNA binding left intact and the RuvC nuclease domain implicated. Reason: The term is true but less specific than the evidence supports: the inhibited enzyme activity is known, so GO:0140721 nuclease inhibitor activity, an is_a descendant of GO:0140678 via GO:0004857 enzyme inhibitor activity, should be used instead. This is the term the equivalent review in this repository uses for AcrIIC1 (genes/NEIME/acrIIC1). I deliberately stop at nuclease inhibitor activity rather than descending to GO:0060703 deoxyribonuclease inhibitor activity: the entity bound and inhibited is an RNA-guided ribonucleoprotein rather than a classical deoxyribonuclease, and the reported target within it is the RuvC domain of that holo-enzyme, so the parent term is the defensible stopping point. Specificity is real and not merely assumed - AcrIIA5 does not inhibit the unrelated RNA-guided nuclease Cas12a. Proposed replacements: nuclease inhibitor activity Supporting Evidence: PMID:32544231 Purified AcrIIA5 completely inhibited target double-stranded DNA (dsDNA) cleavage of Cas9βsgRNA in the in vitro nuclease activity assay, demonstrating that AcrIIA5 alone is sufficient for Cas9 inhibition PMID:32544231 Remarkably, AcrIIA5Ξ20 completely lost its ability to inhibit Cas9, revealing that the N-terminal disordered region is essential for Acr activity PMID:32544231 AcrIIA5 did not inhibit L. bacterium Cas12a, another RNA-guided endonuclease from the type V-A CRISPRβCas system PMID:31775029 AcrIIA5 inhibits Cas9 by preventing DNA target cleavage, but DNA target binding of Cas9 is unaffected. |
| GO:0030430 host cell cytoplasm | IC PMID:28785032 An anti-CRISPR from a virulent streptococcal phage inhibits ... | NEW | Summary: AcrIIA5 is encoded by a strictly lytic (virulent) phage and acts on the bacterial host's Cas9 ribonucleoprotein, a cytoplasmic enzyme, during infection. Inhibition is demonstrated inside living cells of the native host genus, of a heterologous bacterium and of human cells expressing Cas9, so the protein must reach the compartment in which Cas9 resides. No localisation experiment has been performed; the NMR structure and all biochemistry were done in vitro, so this is a curator inference. Reason: This entry is assigned to the phage taxon (NCBITaxon:2006928), and because the phage is virulent rather than a resident prophage, the cell whose cytoplasm the protein occupies is a separate host cell. GO:0030430 host cell cytoplasm is therefore the correct component term, which is where this case differs from genes/LISMN/acrIIA4: that entry is filed under the Listeria monocytogenes taxon because the gene lies in a resident prophage, so GO:0005737 cytoplasm applies there. Evidence is IC rather than IDA because the location is inferred from the target and from in-cell activity, not measured. Added conservatively as the single cellular-component claim; no sub-compartment or membrane association is implied. Supporting Evidence: PMID:28785032 we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus PMID:30046034 we demonstrate that both Acr families from S. thermophilus phages inhibit Cas9-mediated genome editing of human cells |
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Download this section (compressed HTML)Q: Does the disordered N-terminal tail of AcrIIA5 occlude the RuvC active site directly, or does it act allosterically by mispositioning the guide RNA, as the authors of the structural work leave open?
Q: Why is D4276 AcrIIA5 only weakly active against St3Cas9 when the 87% identical D1126 orthologue inhibits it completely, and which substitutions account for the difference?
Q: Should GO offer a term for inhibition that leaves substrate binding intact, given that AcrIIA5 is reported to be unique among Cas9 inhibitors in not competing with target DNA, and that nuclease inhibitor activity does not distinguish this case from the DNA-mimicry inhibitors?
Q: Is the guide-RNA cleavage that accompanies AcrIIA5 activity part of the inhibitory mechanism in vivo, or a downstream consequence of a destabilised Cas9-guide complex?
Experiment: Determine a structure of the AcrIIA5-Cas9-sgRNA complex, by cryo-EM or by NMR with selective labelling of the tail, to locate the disordered region on Cas9 and test the direct occlusion versus allosteric models.
Experiment: Assay target-strand and non-target-strand nicking separately, using Cas9 nickase variants and strand-specific substrates, to establish which cleavage event AcrIIA5 blocks and whether the block is confined to RuvC.
Experiment: Swap the divergent residues between the D4276 and D1126 orthologues and assay each chimera against St1Cas9, St3Cas9 and SpCas9, to map the determinants of orthologue specificity.
Experiment: Co-express AcrIIA5 with Cas9 in hosts individually depleted of candidate ribonucleases, measuring guide-RNA integrity and cleavage inhibition in parallel, to test whether guide degradation is required for inhibition.
Experiment: Measure AcrIIA5 abundance and Cas9 occupancy through a natural phage D4276 infection time course of an immunised Streptococcus thermophilus host, to establish whether the protein accumulates fast enough to outrun target cleavage in the native setting.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: How the disordered tail of AcrIIA5 actually prevents DNA cleavage is unknown. Whether its positive charges occlude the RuvC active site directly or displace the guide RNA and the scissile strand allosterically has not been determined, and no AcrIIA5-Cas9 complex structure exists.
OPEN BIOLOGY
What is known: Established: AcrIIA5 binds only guide-loaded Cas9, does not compete with target-DNA binding, needs more than 20 residues of disordered tail and the Arg12/Lys13/Arg14 charge cluster, and RuvC rather than HNH is the affected domain. Unknown: the contact surface on Cas9 and the physical basis of the block.
Significance: It is the one Cas9 inhibitor reported to act without competing for the DNA target, so its mechanism is not reducible to the DNA-mimicry or HNH-blockade paradigms and would define a third class of anti-CRISPR mechanism.
What would resolve it: A structure or crosslinking map of the AcrIIA5-Cas9-sgRNA complex, with the tail resolved, together with cleavage assays that separate target-strand from non-target-strand nicking.
Provenance (the field's own admissions):
Gap: Whether the partial guide-RNA cleavage seen whenever AcrIIA5 is co-expressed with Cas9 is a mechanistically necessary consequence of inhibition or an incidental effect of host ribonucleases acting on a destabilised complex has not been tested directly.
OPEN BIOLOGY
What is known: Established: the cleavage occurs with several AcrIIA5 homologues and two different Cas9 orthologues, is absent with inhibition-dead AcrIIA5 mutants, and purified AcrIIA5 has no ribonuclease activity of its own. Unknown: whether guide cleavage contributes to inhibition in vivo or merely reports on it.
Significance: If guide degradation contributes to inhibition, AcrIIA5 is effectively irreversible in vivo in a way the in vitro assays cannot show, which matters both for phage biology and for its use as an off-switch.
What would resolve it: Repeat the co-expression experiments in hosts depleted of candidate ribonucleases, and measure guide integrity and cleavage inhibition in parallel over an infection time course.
Provenance (the field's own admissions):
Gap: The reason the His66/Asn70/His73 variant inhibits SpCas9 in a phage plaquing assay but fails to inhibit the same enzyme in vitro is unexplained.
OPEN BIOLOGY
What is known: Both results are reported and each is internally controlled; the discrepancy is acknowledged but not resolved by either group.
Significance: These residues sit in the mobile loop that the structural work implicates in inhibition, so the discrepancy may point to a host factor, a concentration regime or a conformational state present in the cell and absent from the reconstituted assay.
What would resolve it: Titrate the variant against Cas9 over a wide concentration range in vitro and measure its intracellular abundance and Cas9 occupancy in the plaquing host.
Provenance (the field's own admissions):
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