JBD30_035 is the 78-residue product of gene 35 of Pseudomonas phage JBD30 and is the founding type I-F anti-CRISPR protein, AcrF1 (AcrIF1). It is a small globular protein built from a four-stranded antiparallel beta-sheet flanked by two alpha-helices, and it is classified by sequence in the AcrIF1 family (CDD cd22279, Pfam PF20829). Inside an infected Pseudomonas aeruginosa cell it binds the Cas7f (Csy3) backbone of the host type I-F crRNA-guided surveillance complex, two copies per complex contacting four of the six Cas7f subunits, and sits over the Cas7f thumbs so that a target DNA strand cannot move between successive complementary segments of the crRNA guide. The result is a stoichiometric, steric block on target-DNA recognition that leaves crRNA levels and cas gene expression untouched. Because the same surveillance complex is also required for new spacer uptake in this subtype, the protein blocks CRISPR adaptation as well as interference. Loss of the gene makes the phage unable to grow on a CRISPR-proficient host while leaving growth on a CRISPR-deleted host unaffected, and transferring the gene into a CRISPR-sensitive phage lets that phage escape immunity, so the protein's biological role is to neutralise host CRISPR-Cas immunity against the infecting phage genome. It has no catalytic activity and acts as an inhibitory binding partner of a host ribonucleoprotein.
Definition: A molecular function that binds a CRISPR-Cas crRNA-guided surveillance complex and prevents it from recognising or engaging its nucleic acid target, thereby inhibiting CRISPR-Cas-mediated immunity. Includes anti-CRISPR proteins that occlude the target-DNA path on the complex sterically and those that prevent target engagement by other means, and excludes inhibitors acting on a separate nuclease or helicase recruited by the complex.
Justification: GO has a biological-process term for anti-CRISPR action (GO:0098672 symbiont-mediated suppression of host CRISPR-cas system) but no molecular-function term for the activity that carries it out. The only available options are uninformative binding terms, so the molecular layer of an entire and well-characterised protein class, including AcrF1, AcrF2 and AcrF8 against the type I-F Csy complex and AcrIIA4 and AcrIIA5 against Cas9, is unrepresentable. The activity is a specific, assayable inhibition of a defined ribonucleoprotein machine, and a molecular-function term would also let the existing process rows be grounded in a mechanism rather than restated.
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0098672 symbiont-mediated suppression of host CRISPR-cas system | IMP PMID:26586803 Foreign DNA acquisition by the I-F CRISPR-Cas system require... | ACCEPT | Summary: CACAO row (assigned_by CACAO, dated 2026-02-16), the only GO annotation this entry carries. What the cited paper shows for this gene: Vorontsova et al. reconstituted the Pseudomonas aeruginosa type I-F CRISPR-Cas system in Escherichia coli from pCas and pCsy plasmids, expressed each of the five known anti-CRISPR proteins from a compatible plasmid, and found that spacer acquisition was abolished by every one of them, including acrF1, which is the product of this entry. The control that carries the annotation is a frame-shift allele of acrF1: it lost the anti-acquisition activity just as it had previously lost anti-interference activity, which is why the row is IMP rather than IDA. The term matches: GO:0098672 is defined as a symbiont inhibiting or disrupting the CRISPR-Cas system of its host and is the most specific term GO offers (QuickGO 2026-11-01: non-obsolete, no children, usage unrestricted), its own definition is cited to this protein's discovery and mechanism papers (PMID:23242138, PMID:26416740), and the paper measures suppression of the host system directly rather than a downstream phenotype. Two evidential caveats that do not change the judgement: the assay is a heterologous reconstitution in E. coli rather than the natural PA14 host, and the arm of the system measured is adaptation rather than interference, which the generic term covers. Reason: The claim is correct, maximally specific for this gene and directly supported by the cited experiment, and it is independently established in the natural host: deleting or frame-shifting gene 35 renders JBD30 unable to replicate on wild-type PA14 while leaving replication on the CRISPR-deleted strain intact (PMID:23242138). 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, or whose participation is unknown; here the mechanism is settled. AcrF1 binds the Cas7f (Csy3) backbone of the host surveillance complex and occludes the target-DNA path (PMID:26416740, PMID:28340349), so the protein physically performs the suppression and involved_in is the correct relation. This is a source-record field error, recorded in source_defects, and not a reason to reject the claim, downgrade it, or change the term, reference or evidence code. The hedge would have been defensible had the paper shown only that the gene is required for anti-CRISPR function without identifying what the product acts on, which is not the case. Supporting Evidence: PMID:26586803 Spacer acquisition was inhibited in the presence of each of the five different anti-CRISPR proteins tested (acrF1β5, Figure 4). PMID:26586803 A plasmid bearing a frame-shift mutation in the beginning of the acrF1 anti-CRISPR gene that inactivates its anti-interference function (40) also inactivated the anti-acquisition function. PMID:26586803 We conclude that anti-CRISPR proteins that inhibit CRISPR interference in P. aeruginosa by targeting either the Csy complex or Cas3, also inhibit spacer acquisition. PMID:23242138 a frameshift mutation was introduced into the phage JBD30 anti-CRISPR gene (gene 35). This mutant phage was unable to replicate on wild-type PA14 but still replicated robustly on the ΞCR/cas strain, demonstrating the requirement of the anti-CRISPR gene for replication PMID:28340349 AcrF1 binds to the Cas7f backbone and prevents target binding |
| GO:0043021 ribonucleoprotein complex binding | IPI PMID:28340349 Structure Reveals Mechanisms of Viral Suppressors that Inter... | NEW | Summary: Proposed new annotation. This entry has no molecular-function annotation at all, yet its activity is one of the better characterised in the anti-CRISPR field: it binds the crRNA-loaded type I-F Csy surveillance complex, a ribonucleoprotein, through the Cas7f (Csy3) protein subunits. The interaction is shown by co-purification and binding kinetics (PMID:26416740) and resolved in the cryo-EM structure of the Csy complex with two AcrF1 copies contacting four of the six Cas7f subunits, with surface-plasmon dissociation rates for wild-type and Cas7f K85A complexes (PMID:28340349). The supporting entity is Pseudomonas aeruginosa UCBPP-PA14 Csy3/Cas7f, UniProtKB:Q02MM1 (reviewed in this repository as genes/PSEAB/csy3). Reason: GO has no molecular-function term for anti-CRISPR activity (a QuickGO text search on 2026-11-01 returns only BP terms plus unrelated anti-sigma and anti-Mullerian terms), so the informative existing term for what this protein does is binding of the host surveillance ribonucleoprotein; bare protein binding would be uninformative and is avoided. This follows the precedent set for the homologous Csy-complex inhibitor AcrF8 (genes/BPZF4/AcrF8), whose core molecular function is recorded with the same term, and it is the activity that the accepted biological-process row rests on, so it adds the missing mechanistic layer rather than a second claim about the same thing. A mechanism-specific molecular-function term is filed under proposed_new_terms. Supporting Evidence: PMID:28340349 The structure presented here explains how two AcrF1 proteins (i.e. AcrF1.1 and 1.2) bind to one Csy complex through interactions with four of the six Cas7f subunits PMID:28340349 AcrF1 proteins sits on top of the Cas7.6f and Cas7.4f thumbs, blocking the transition of target DNA from 1 to 2 and segment 3 to 4, respectively. PMID:26416740 Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. |
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Download this section (compressed HTML)Q: Is the inward displacement of the Cas7.4f and Cas7.6f web domains in the AcrF1-bound Csy complex caused by AcrF1, making it an allosteric as well as a steric inhibitor?
Suggested experts: Blake Wiedenheft, Gabriel C. Lander
Q: Does AcrF1 block primed and naive spacer acquisition equally, and does it contact Cas1 or Cas2-3 at all, or is occupancy of the Csy complex the whole explanation for its anti-adaptation effect?
Suggested experts: Konstantin Severinov, Ekaterina Semenova
Q: Cas7f lysines K85, K254 and K257 are required for AcrF1 binding but mutating them also cripples target-DNA binding. Does that constraint explain why type I-F hosts appear not to escape AcrF1 by point mutation, and what route does escape take instead?
Suggested experts: Alan R. Davidson, Karen L. Maxwell
Experiment: Determine a cryo-EM structure of the apo Pseudomonas aeruginosa Csy complex to the same resolution as the AcrF1/AcrF2-bound complex and compare web-domain positions subunit by subunit, with an orthogonal solution readout (site-specific FRET or crosslinking across the web-to-crRNA gap) measured with and without saturating AcrF1. Identical apo and bound web positions would establish the purely steric model; a shift that tracks AcrF1 occupancy would define a second, allosteric contribution.
Hypothesis: AcrF1 inhibits the Csy complex purely by steric occlusion of the target-strand path, not by an allosteric change in the Cas7f web domains.
Type: comparative cryo-EM with solution conformational reporters
Experiment: Reconstitute type I-F adaptation and titrate AcrF1 against separately purified Csy complex, Cas1 and Cas2-3, measuring binding by pull-down, SPR and crosslinking mass spectrometry; in parallel measure naive and primed acquisition in cells expressing AcrF1 at graded levels and in cells carrying a Cas7f variant that cannot bind AcrF1 but retains function. Proportional loss of acquisition with Csy occupancy, and no detectable AcrF1-Cas1 or AcrF1-Cas2-3 complex, would confirm the single-target model.
Hypothesis: AcrF1's block on spacer acquisition is entirely a consequence of occupying the Csy complex, with no direct engagement of the adaptation machinery.
Type: reconstituted adaptation assay with quantitative interaction mapping
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether AcrF1 binding is itself responsible for the inward shift of the Cas7f web domains seen in the Csy-AcrF1-AcrF2 structure, and so whether the block on target DNA is purely steric or partly conformational, is unresolved, because no structure of the Csy complex free of anti-CRISPR proteins was available for comparison.
OPEN BIOLOGY
What is known: Established: two AcrF1 copies bind four of six Cas7f subunits, contact the conserved thumb lysine K85, dissociate ~10^3-fold more slowly from wild-type than from K85A complexes, and sit where the target strand must cross between guide segments. Unknown: whether the adjacent web-domain displacement is caused by AcrF1 or is an intrinsic feature of the complex.
Significance: It decides whether AcrF1 is a pure steric occluder, the paradigm it is usually cited as, or an allosteric inhibitor, which would change how escape mutations and engineered variants are predicted.
What would resolve it: A cryo-EM structure of the apo Csy complex at comparable resolution, compared subunit by subunit with the AcrF1-bound complex, together with crosslinking or FRET reporters of the web domain position in the presence and absence of AcrF1.
Provenance (the field's own admissions):
Gap: Why AcrF1 blocks new spacer acquisition is explained by the host genetics rather than by anything measured on AcrF1 itself: the Csy complex is required for adaptation, so inactivating it is sufficient. Whether AcrF1 additionally contacts Cas1, Cas2-3 or the adaptation intermediate, and whether it blocks naive and primed acquisition equally, has not been tested.
OPEN BIOLOGY
What is known: Established: expression of acrF1 abolishes spacer acquisition in a heterologous reconstitution, and a frame-shift allele does not. Unknown: whether the effect is wholly accounted for by occupancy of the Csy complex, or whether AcrF1 engages the adaptation machinery directly.
Significance: Anti-CRISPRs are increasingly deployed as off-switches for engineered CRISPR systems, and an inhibitor that acts on both arms through one contact is a different tool from one with two independent targets.
What would resolve it: Pull-downs and crosslinking of AcrF1 against Cas1, Cas2-3 and the Csy complex in the presence and absence of a protospacer substrate, with acquisition assays comparing naive and primed adaptation at AcrF1 concentrations that partially occupy the Csy complex.
Provenance (the field's own admissions):
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