Csy1, also called Cas8f, is the large subunit of the Csy crRNA-guided surveillance complex of the Pseudomonas aeruginosa UCBPP-PA14 type I-F (Yersinia-subtype) CRISPR-Cas system. The complex comprises one Cas8f/Csy1, one Cas5f/Csy2, six Cas7f/Csy3 and one Cas6f/Csy4 wrapped around a single 60-nucleotide crRNA in a helically twisted G shape; Csy1 occupies the PAM-proximal tail together with Cas5f. It has three assignable jobs there. Its N-terminal hook domain, lined with basic residues, forms the pocket that grips the protospacer-adjacent-motif-containing target DNA duplex; a central loop known as the lysine wedge inserts into the duplex fork, reads the motif through minor-groove contacts and splits the strands so the crRNA can base pair with the target strand; and together with Cas5f it clamps the sequence-specific 5' handle of the crRNA that sets the guide register. Completion of base pairing along the whole guide then licenses recruitment of the Cas3 helicase-nuclease to the Csy1 C-terminal helical bundle, the step at which the phage anti-CRISPR protein AcrIF3 intervenes from the Cas3 side.
Definition: A ribonucleoprotein complex composed of CRISPR-associated (Cas) proteins assembled around a single mature CRISPR RNA (crRNA), which uses the crRNA as a guide to locate nucleic acid complementary to its spacer sequence. In type I systems the complex additionally recognises a protospacer-adjacent motif, forms an R-loop at the target, and recruits a trans-acting nuclease for target destruction. The Pseudomonas aeruginosa type I-F complex, Csy, has the composition Cas8f(1)/Cas5f(1)/Cas7f(6)/Cas6f(1) with one 60-nucleotide crRNA; the type I-E equivalent is Cascade.
Justification: GO's entire CRISPR vocabulary is five biological-process terms (GO:0099048, GO:0043571, GO:0098672 and the obsolete GO:0110132 and GO:0110133), with no cellular-component term, so every subunit of every CRISPR surveillance complex can currently only be placed on the bare GO:0032991 root or on its generic child GO:1990904. The complex is a well-defined, stoichiometrically determined, structurally solved entity that ComplexPortal already models for the type I-E case (CPX-1005, Cascade complex), and the Cas7f backbone in particular is the surface targeted by several families of phage-encoded anti-CRISPR proteins, so a specific term would make a large body of structural and counter-defence curation expressible. This is the same request made from the Escherichia coli type I-E Cascade subunit reviews, so that the two systems ask for one term rather than two.
Parent term: ribonucleoprotein complex
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | MODIFY | Summary: Uninformative as a molecular function. The Csy1-Csy2 interaction records a contact made inside the Csy ribonucleoprotein surveillance complex. Cas8f/Csy1 and Cas5f/Csy2 together build the PAM-proximal tail of the spiral: the two subunits jointly clamp the 5' handle of the crRNA into its bent conformation, and the Cas8f hook and the Cas5f thumb form two of the three walls of the pocket that holds the target duplex. The contribution is therefore structural, and it is mutual. Reason: The term "protein binding" conveys nothing about function. The underlying observation is a contact internal to the Csy surveillance complex, so the honest subunit-level function is GO:0005198 structural molecule activity, defined as the action of a molecule that contributes to the structural integrity of a complex. None of its 22 children fits a non-ribosomal ribonucleoprotein, so the parent is the correct level. This keeps the replacement in the molecular-function aspect, which the GOA row's enables qualifier requires: a gene product cannot enable a cellular component, so the complex-membership reading of this row, however well supported, cannot be expressed by replacing an enables MF term with a CC term. Complex membership is instead carried by a dedicated NEW part_of GO:1990904 ribonucleoprotein complex row and by core_functions in_complex, and the missing specific term is requested in proposed_new_terms. The same replacement is used for the equivalent intra-complex rows on cas6f, csy1, csy2 and csy3, and matches how the five Escherichia coli type I-E Cascade subunits were treated. Proposed replacements: structural molecule activity Supporting Evidence: PMID:32170016 the Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle PMID:28985564 the duplex region of the target DNA (including the G-C/G-C PAM) is sandwiched between the Cas8f N-terminal hook domain (residues 1-166), the Cas5f thumb domain (residues 48-109), and Cas7 |
| GO:0005515 protein binding | IPI PMID:26416740 Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR... | MODIFY | Summary: Second IntAct record of the same Csy1-Csy2 contact, from the study of anti-CRISPR mechanisms. Treated identically to the PMID:21536913 row: what the interaction shows is this subunit's structural contribution to the surveillance complex, not a binding activity. Reason: Consistency with the other protein binding row on this protein and with the four Csy subunits and the five Escherichia coli Cascade subunits; the paper's subject is inhibition of the assembled complex, which presupposes the structural contribution being asserted. GO:0005198 keeps the replacement in the molecular-function aspect that the enables qualifier requires. Proposed replacements: structural molecule activity Supporting Evidence: PMID:26416740 Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits PMID:32170016 The Csy complex is comprised of four types of Cas proteins (Cas5f-8f) and a single 60-nt crRNA |
| GO:0003677 DNA binding | IDA PMID:28985564 Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Ta... | NEW | Summary: Cas8f supplies the dominant protein contacts to the target DNA duplex. Cryo-EM of the Pseudomonas aeruginosa Csy complex bound to a PAM-containing double-stranded target shows the duplex clamped by the Cas8f N-terminal hook, whose basic residues line the binding pocket, and a Cas8f loop wedged into the duplex fork making minor-groove contacts to the motif bases. Functional confirmation comes from the wedge residue K247, shown to be required for the duplex splitting that precedes DNA binding by the complex. Reason: Missing from GOA, which holds only two protein binding rows for this protein. Comparator check: the type I-E large subunit casA/Cse1 (UniProtKB:Q46901) carries GO:0003677 by IDA from PMID:25123481, so a DNA-binding term is the convention for this subunit position. Cas8f is the entity making the contacts, not merely a subunit of something that does. Supporting Evidence: PMID:28985564 the duplex region of the target DNA (including the G-C/G-C PAM) is sandwiched between the Cas8f N-terminal hook domain (residues 1-166), the Cas5f thumb domain (residues 48-109), and Cas7 PMID:28985564 Numerous positively charged residues line this DNA binding pocket, including residues from Cas7.6f (K299), the Cas5f thumb (R90), and especially the Cas8f hook (R24, K28, K31, R59, K71 and R78) PMID:32170016 Residue K247 of Cas8f has been shown to promote foreign DNA duplex splitting by wedging into the strands, which is a prerequisite for DNA binding to Csy |
| GO:0003723 RNA binding | IDA PMID:32170016 Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against typ... | NEW | Summary: Cas8f, with Cas5f, clamps the 5' handle of the crRNA, and the contacts are base-specific rather than backbone-only, which is what fixes the register of the guide within the complex. Reason: Missing from GOA. Comparator check: casA/Cse1 (Q46901) carries GO:0003723 by IDA twice, and casC, casD and casE carry it too, so RNA binding is annotated across the Cascade subunits. Here the sequence-specific 5'-handle recognition is directly resolved in the structure of this complex. Supporting Evidence: PMID:32170016 the Cas5f and Cas8f subunits in the tail region also make extensive interactions with crRNA, causing the 8 nt of crRNA at its 5′ terminus to display an “S”-shape architecture, called the 5′ handle PMID:32170016 supporting a sequence-specific recognition of the crRNA 5′ handle by Cas5f and Cas8f |
| GO:0099048 CRISPR-cas system | IDA PMID:28985564 Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Ta... | NEW | Summary: Cas8f does work in the target interference stage named in this term's definition: it reads the protospacer-adjacent motif and splits the duplex, which are steps of target recognition rather than preconditions for it. Reason: Comparator check: all five Escherichia coli Cascade subunits carry involved_in GO:0099048 by IDA from PMID:18703739, yet no Pseudomonas aeruginosa PA14 Csy subunit does, which marks a per-strain curation lag rather than a convention about structural subunits. Checked against QuickGO: GO:0099048 is neither an ancestor nor a descendant of GO:0043571 or GO:0051607, so nothing is duplicated. Supporting Evidence: PMID:28985564 Wedged into this fork is the tip of a loop (residues 246-250, sequence TKPQN) emanating from the central domain of Cas8f PMID:28985564 The placement of this loop, which we refer to as the lysine-containing wedge (or K-wedge), would sterically block a 1TS-1NTS base pair PMID:21536913 the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences |
| GO:1990904 ribonucleoprotein complex | IDA PMID:21536913 RNA-guided complex from a bacterial immune system enhances t... | NEW | Summary: Cas8f/Csy1 is an integral subunit of the Csy surveillance complex, which forms the PAM-proximal tail together with Cas5f/Csy2. The complex contains a single mature 60-nucleotide crRNA as an integral component, not as a ligand, so it is a ribonucleoprotein complex rather than merely a protein-containing one. Reason: Complex membership is not currently annotated for any Pseudomonas aeruginosa PA14 Csy subunit, whereas all five Escherichia coli type I-E Cascade subunits (casA, casB, casC, casD, casE) carry a part_of protein-complex row by IDA or IPI. The GOA rows that record this contact for the PA14 proteins carry the enables qualifier and are therefore molecular-function rows, which cannot be converted to a cellular-component term; the membership claim consequently needs its own row, which is what this is. GO:1990904 is used rather than the bare GO:0032991 root because the guide RNA is an integral component of the assembly, and because GO has no cellular-component term for any CRISPR surveillance complex - its entire CRISPR vocabulary is five biological-process terms - so GO:1990904 is the most specific existing term. The missing specific term is requested in proposed_new_terms. Supporting Evidence: PMID:21536913 the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences PMID:32170016 the maps exhibit an asymmetric spiral, with one Cas6f subunit in the head, one Cas5f and one Cas8f in the tail, and six Cas7f comprising the spiral backbone PMID:32170016 The Csy complex is comprised of four types of Cas proteins (Cas5f-8f) and a single 60-nt crRNA |
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Download this section (compressed HTML)Q: GO has no term for crRNA-guided target recognition. Should such a term be placed on the surveillance complex as a whole, with subunits as part_of, or should individual subunits carry contributes_to? Cas8f is the hardest case, because PAM reading is crRNA-independent while the rest of recognition is not.
Q: Which Cas8f surface does Cas3 engage, and is the recruitment signal a specific conformational change in the C-terminal bundle or the global helical elongation of the complex on target binding?
Q: PA14 disruption of csy1 lowers but does not abolish crRNA, while disruption of csy2 abolishes it. Does Cas8f protect the 5' handle from degradation only redundantly with Cas5f, or does it act later, at loading rather than at stabilisation?
Experiment: Build PA14 strains carrying Cas8f K247A, Q249A and N250A substitutions, confirm Csy complex assembly and crRNA loading by pull-down and northern blot, then measure plasmid-targeting and phage-restriction efficiency. This separates the PAM-reading function from complex integrity.
Hypothesis: The Cas8f lysine wedge is required for target binding in vivo as well as for duplex splitting in vitro, and a wedge mutant produces a complex that is assembly competent but interference dead.
Type: structure-guided mutagenesis with in vivo targeting assay
Experiment: Crosslink and mass-spectrometry map the Cas8f-Cas3 interface on the target-bound complex, then test candidate interface substitutions for loss of Cas3-dependent DNA degradation in the reconstituted assay while retaining target binding.
Hypothesis: Cas3 is recruited through the Cas8f C-terminal helical bundle, and masking that bundle phenocopies AcrF3.
Type: crosslinking mass spectrometry with reconstituted cleavage assay
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: GO has no molecular-function term for crRNA-guided, PAM-licensed target recognition, which is the activity the assembled Csy complex has and which Cas8f licenses. The component activities that can be annotated (DNA binding, RNA binding) do not distinguish this protein from any unguided nucleic-acid-binding protein.
OPEN ONTOLOGY
What is known: The chemistry on either side is covered: the nuclease activities of Cas3 and Cas6f have terms, and the structural steps (PAM reading, duplex splitting, R-loop formation, directional target verification) are resolved to near-atomic detail. What is missing is a term for the guide dependence that defines the system.
Significance: Without such a term the defining property of every Cas effector is invisible to GO-based reasoning, and the host target of a whole class of phage anti-CRISPR proteins cannot be stated.
What would resolve it: Request a molecular-function term for guide-RNA-dependent nucleic acid recognition, with children for RNA-guided DNA endonuclease activity and RNA-guided RNA endonuclease activity, as proposed in modules/crispr_cas_adaptive_immunity.yaml. No existing id has been substituted for the missing concept here.
Provenance (the field's own admissions):
Gap: How Cas8f signals a verified target to Cas3 is undetermined. The docking site is placed on the Cas8f C-terminal helical bundle only by inference, and no structure of a Cas8f-Cas3 interface exists for this system.
OPEN BIOLOGY
What is known: Established: R-loop completion is the trigger, Cas3 is the recruited nuclease, and the AcrF3 structure shows which Cas3 surfaces are masked to prevent recruitment. Not established: the reciprocal Cas8f surface, and whether the large helical elongation the complex undergoes on DNA binding is itself the recruitment signal.
Significance: This is the committed step of type I-F interference and the step one of the best-characterised anti-CRISPR proteins blocks; the host side of that inhibition cannot be modelled without it.
What would resolve it: Determine a structure of the target-bound Csy complex with Cas3 engaged, and map the interface genetically with Cas8f C-terminal bundle substitutions.
Provenance (the field's own admissions):
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