Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4

Domain: biology
Published: 2025-12-15
Editorial outcome: major_revision

Abstract
Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.

Introduction
CRISPR-Cas systems provide adaptive immunity against invading mobile genetic elements (MGE) in prokaryotes1. Type III CRISPR-Cas systems comprise a ribonucleoprotein effector complex programmed with CRISPR RNA (crRNA) to detect invading RNA from viruses and other MGE2. Target RNA binding activates the catalytic Cas10 subunit, which in approximately 90% of cases harbours a specialised polymerase domain for the synthesis of cyclic oligoadenylate (cOA) molecules3–6. cOA acts as a second messenger, signalling viral infection in the cell by binding and activating a wide range of ancillary defence proteins (reviewed in ref. 7). Cleavage of bound target RNA deactivates the Cas10 cyclase activity8 and extant cOA molecules are degraded by enzymes known as ring nucleases9.

The Csm6/Csx1 family of type III CRISPR ancillary effectors use a dimeric CARF domain for cOA binding, resulting in allosteric activation of their HEPN-family ribonuclease domains5,6. Some of these effectors also degrade cA4 or cA6 in their CARF domains, providing a mechanism for auto-deactivation of the CRISPR immune response10–14. The effectors Cami1 and CalpL, which detect cA4 via CARF and SAVED domains, respectively, also possess intrinsic ring nuclease activity15–17.

The first extrinsic ring nuclease to be described, Crn1, was purified from Saccharolobus solfataricus, revealing a small protein with a dimeric CARF domain for cOA recognition. Crn1 slowly degrades cyclic tetra-adenylate (cA4) to linear di-adenylate products18. A second family of extrinsic ring nucleases, Crn2, use a dimeric domain unrelated to CARF proteins to bind and degrade cA4. Crn2 is found both in CRISPR defence operons19–21 and in viral genomes, where it functions as an anti-CRISPR (Acr) by rapidly degrading cA4 to neutralise cellular defences21. A third ring nuclease family, Crn3, uses a fold related to CARF domains to sandwich cA4 in protein tetramers, degrading the signalling molecule in a metal-dependent reaction to linear di-adenylates22,23. Finally, a group of related proteins (Csx15, Csx16 and Csx20) were predicted to act as ring nucleases24. Recently, we demonstrated that Csx16 and Csx20 are cA4-specific ring nucleases, and showed that the majority of cA4- and cA6-signalling type III CRISPR systems include a means to degrade their activator25. This has important implications for cellular outcomes on viral infection, as it provides a means to avoid programmed cell death or growth arrest25,26.

We previously reported a systematic bioinformatic analysis of type III CRISPR systems, which resulted in the identification of a new effector protein, Csm6-2, which is activated by cyclic hexa-adenylate (cA6) and cleaves RNA non-specifically3. Csm6-2 is typically found associated with a CorA-family trans-membrane effector, and in some instances, the two proteins are fused. The CRISPR-associated CorA effector from Bacteroides fragilis was previously shown to be activated by SAM-AMP—a signalling molecule derived from conjugation of ATP and S-adenosylmethionine27, but systems encoding both Csm6-2 and CorA may signal via cA63.

Here, we show that these CRISPR loci include a small open reading frame encoding a hypothetical protein, hereafter named Crn4 (CRISPR ring nuclease 4), which degrades cA3, cA4 and cA6 to a range of linear products. The crystal structure of Crn4 reveals a novel, dimeric fold with conserved histidine and arginine residues that are essential for catalysis. Crn4 can partially relieve CRISPR-mediated immunity in a plasmid challenge assay, consistent with a function as a cellular ring nuclease, and is encoded in some phage and plasmid genomes where it likely functions as an Acr.

Results
Identification of the CRISPR associated protein Crn4
Csm6-2, a fused Csm6 orthologue that is activated by cA6 and cleaves RNA, was recently identified from a bioinformatic analysis of type III CRISPR loci3. A systematic scan of the regions neighbouring Csm6-2 revealed a small open reading frame (ORF) encoding a hypothetical protein of unknown function in 12 CRISPR type III-D loci. In 5 of the 12 instances, a CorA membrane effector was also present (Fig. 1a). In two cases, the ORF was fused with Csm6-2, and in one instance it was fused with the effector TIR-SAVED (Fig. 1b)3,28. A recent bioinformatic study identified this ORF, named Unk01, as a potential ring nuclease24.

Using hidden Markov model (HMM) profiles based on these 12 homologues, we identified 10 additional homologues in our dataset, which were associated with the cA4-activated effector Can2, and two homologues with no recognisable effector in the CRISPR locus. Phylogenetic analysis divided the 22 homologues into two distinct groups: group “a” associated with Csm6-2/CorA, and group “b” associated with Can229,30 or lacking a recognizable effector (Fig. 1a). The small size of the predicted proteins, combined with their consistent association with cA4 and cA6 activated effectors (either through adjacency or fusion) and prevalence on MGEs supported the prediction that Unk01 was a ring nuclease. Following established nomenclature, we provisionally designated this family of proteins CRISPR ring nuclease 4 (Crn4). The family was sub-divided into Crn4a and Crn4b, distinguishable as Crn4b members are smaller, lacking a ~10 amino acid region present in Crn4a (Supplementary Fig. 1). Crn4b is also observed in the genomes of phages and plasmids. Of the 28,137 phage genomes in the Millard database31, 11 contained a Crn4 homologue, while this was true for 10 out of 59,895 plasmid sequences in the database PLSDB32. Some of the phage and plasmid orthologues have an 60–80 amino acid N-terminal extension of unknown function (Supplementary Fig. 2). All members of the family have conserved residues including T13, H15 and R112 (A. procaprae Crn4a numbering), which are implicated in cOA binding and catalysis (further details below).

Crn4a is a ring nuclease with broad specificity
We chose candidate Crn4a protein WP_136192672, found in the Actinomyces procaprae type III CRISPR locus adjacent to the previously characterised Csm6-2 protein3, for further study. To explore its function, we constructed a synthetic gene encoding the protein for expression in Escherichia coli and purified it by immobilised metal affinity and size exclusion chromatography (Supplementary Fig. 3).

Crn4a was tested for ring nuclease activity by incubating the purified protein with synthetic cOA species under multiple turnover (substrate excess) conditions in EDTA, followed by HPLC analysis (Fig. 2a). Crn4a cleaved all three cOA species tested, cA6, cA4 and cA3, generating linear products. cA6 was degraded with a rate constant of 0.024±0.007 min−1 (Fig. 2b and Supplementary Fig. 4), cA3 slightly faster (0.083±0.02 min−1) and cA4 was degraded much more rapidly (0.98±0.1 min−1). cA4 and cA6 were cleaved on both sides of the ring, yielding linear 2′,3′-cyclic phosphate cA2>P and cA3>P products, respectively (Fig. 2a and Supplementary Fig. 4). This suggests concerted or sequential cleavage of cOA on opposite sides of the molecules, reminiscent of the viral ring nuclease AcrIII-121. cA3, which unlike cA4 and cA6 lacks an axis of two fold symmetry, was degraded to A2>P and A>P, consistent with asymmetric cleavage (Fig. 2a and Supplementary Fig. 4). The ability to cleave multiple cOA species was not observed for Crn1-318,21,22. Thus, in contrast to all other CRISPR associated ring nucleases tested to date, Crn4a has broad specificity for cOA species and is the first confirmed CRISPR-associated cA3 ring nuclease.

We also cloned and expressed the gene encoding a representative of the Crn4b family (MBK8772583 from Hyphomicrobiales bacterium) (Supplementary Figs. 1 and 2). Crn4b degraded cA4 and cA3 but was inactive against cA6 (Fig. 3a). This appears consistent with the association of Crn4b with the Can2 effector, which is activated by cA429,30,33. Although Crn4b produces the same final cA4 cleavage products as Crn4a, intermediate linear products (A2>P and A3>P) were observed at early reaction timepoints with cA4 and cA3, respectively (Fig. 3a and Supplementary Fig. 5), consistent with a sequential cleavage mechanism.

The close association of Crn4a with the Csm6-2 effector prompted us to test the ring nuclease activity of Csm6-2, as all Csm6 proteins studied to date possess intrinsic cA6 ring nuclease activities in the CARF domain of the protein10,11,13. Assays revealed cA6 degradation activity for Csm6-2 (Fig. 3b). The rate of cleavage of cA6, 0.026±0.01 min−1 (Supplementary Fig. 6), was equivalent to that observed for Crn4a. However, neither cA3 nor cA4 were degraded by Csm6-2. This activity could reside in the CARF domain, HEPN domain or a combination of the two.

Given the broad specificity of Crn4a, we also tested its ability to degrade noncanonical substrates (Supplementary Fig. 7). Perhaps unsurprisingly, cA5, which is not known to be an activator of any CRISPR effector, was degraded efficiently by Crn4a. The CBASS signalling molecule cAAG was not a substrate, suggesting a strict specificity for adenine bases, and a high concentration of Crn4 cleaved a 12 nt polyA RNA substrate three nucleotides from the 3′ end. This suggests RNA can fold into the active site and be cleaved by the enzyme. Previously, it was demonstrated that the CARF domain of the Csx1 ribonuclease can bind linear tetra-adenylate RNA tails, which activated the HEPN domain34. The physiological relevance of such observations is questionable, given the slow reaction kinetics.

Structure and mechanism of Crn4
To characterise the structure of Crn4, we crystallised the wild-type Crn4a and Crn4b proteins in apo-form, along with the Crn4a H15A variant in the presence of cA6. We collected X-ray diffraction data to 2.34, 1.09 and 1.44 Å resolution, respectively. The structures were solved using molecular replacement with the AlphaFold predicted structure as the model. The structures reveal a new protein fold, with no hits identified by the DALI server35 with Z-scores higher than 2.6. The core structure comprises four (Crn4b; Fig. 4a) or five (Crn4a; Fig. 4b) pairs of beta strands, one parallel and the others anti-parallel, and one short alpha helix. Both proteins are dimeric, and strikingly, an extended β-hairpin, comprising one (Crn4b) or two (Crn4a) two-stranded anti-parallel beta sheets, from one monomer wraps around the neighbouring subunit, which is largely facilitated by hydrophobic interactions between the two molecules (Fig. 4a, b). The structures of apo Crn4a and Crn4b superimpose with an RSMD of 2.7 Å over 128 Cα residues. The only significant difference is the length of the hairpin loop, which is around 10 residues longer (at the N-terminus of loop) in Crn4a. The loop is unstructured in both proteins, and Crn4a and Crn4b have low sequence conservation in this region (Fig. 4c and Supplementary Fig. 8).

Upon structure solution, there was clear electron density visible in the maximum likelihood/σA weighted Fo-Fc electron density map at 3σ corresponding to a molecule of cA6 bound to Crn4a that originated from the crystals co-crystallised with the ligand (Supplementary Fig. 9A). The structure of Crn4a in complex with cA6 shows the ligand binds at the dimer interface (i.e. one molecule of cA6 to two protein molecules) in a symmetrical compact arrangement, with two adenine bases oriented upwards, two downwards and two roughly axial to the plane of the cA6 ring (Fig. 4d, e and Supplementary Fig. 9B). This differs from the conformation of cA6 bound to Streptococcus thermophilus Csm6, where four of the six adenine bases were in the plane of the ring (Supplementary Fig. 9C)36.

In Crn4a, cA6 hydrogen bonds with the main chain atoms of R92, E16, P77, A70, and side chain hydroxyl of S91, and a phosphate moiety forms an electrostatic interaction with R112 (Supplementary Fig. 9D). In addition, there is pi–pi stacking between an adenine of cA6 and the guanidium group of R92. R92, E16, S91 and R112 are all structurally conserved in Crn4b, as are the main chain atoms of P77 and A70, but with alternative side chains. It is noteworthy that, given the size of cA6, there are relatively few interactions formed with Crn4a, and the majority are with main chain atoms, suggesting the side chain functionality is not particularly important. Interestingly, R92 and R112 (or equivalent) side chains are found in a similar conformation in the two apo structures, but in a different orientation in the cA6 complex with Crn4a, suggesting the residues are flexible and move upon ligand binding. Stabilisation of the negative charge of cA6 is performed by two magnesium ions located in the centre of the cA6. One of these interacts with two adjacent phosphate groups and two water molecules and the other with one phosphate group and three water molecules (Supplementary Fig. 9E).

The orientation of R112 in Crn4a, and its interaction with a phosphate group in cA6, points to this being an important residue in phosphodiester bond cleavage (Fig. 4f). Alignment of the Crn4a H15A mutant (in complex with cA6) and apo Crn4a suggests that His15 would swing into a key position to participate in catalysis. A conserved threonine, T13, is positioned just behind the histidine. It is noteworthy that R112 and H15/T13 are located on different monomers in the dimer. The in-line angles formed by O2′-P-O5′ are between 100 and 110° around the ring, far from the angle of ~160° that is optimal for in-line attack by the 2′-OH nucleophile21. This may reflect the fact that cA6 is not the optimal substrate for this enzyme; additionally, the H15A variant crystallised here may differ from the wild-type enzyme in this respect.

Comparison of the apo and cA6-bound structures of a Crn4a monomer reveals that the globular part overlaps perfectly, but the extended β-hairpin bends upwards at an “elbow” when cA6 is bound (Fig. 4g). In dimeric Crn4a, this movement translates to the protein encasing the cOA upon binding (Supplementary Movies 1 and 2). This conformational change means the loops of the β-hairpin come together to sit above the central cavity where cA6 binds (Supplementary Fig. 9F). H15 and R112 are located on these loops, and move by 6 Å and 7 Å, respectively (Supplementary Fig. 9G), thus placing them in key positions to participate in cOA binding and/or cleavage. As H15 and R112 originate from different monomers in the Crn4a dimer, the precise loop movements from both are critical to form the active site, allowing catalysis to occur. Given the observation of relatively few interactions between cA6 and Crn4a (Supplementary Fig. 4D), it is possible that the protein residues displayed in the “open” form of the dimer are more important for recognition and binding of cOA. Once the cOA is enclosed in the central cavity of the “closed” dimer there is less need for interactions to guide binding or specificity, and only those important for catalysis, or for correct positioning of catalytic residues, are important.

Despite our best efforts, we were unable to crystallise a ligand-bound complex with Crn4b, or indeed Crn4a in complex with cA3 or cA4. Given the considerable movement observed for Crn4a upon binding of cA6 and our suggestion that specificity may largely be driven by interactions with the ‘open’ form of Crn4, direct comparison of the factors influencing specificity for substrate for each protein was not possible. Given the broad substrate specificity of the enzyme, this remains an important topic for future study.

We constructed and purified variants T13A, H15A and R112A of Crn4a to test their role in catalysis. Mutation of H15 or R112 to alanine abolished cleavage of the preferred substrate, cA4, after incubation at 37°C for 60 min, while the T13A variant retained some activity (Fig. 4). Qualitatively similar data were obtained for cA3 and cA6 cleavage (Supplementary Fig. 10). These data are consistent with a role for H15 as a general acid, protonating the oxyanion leaving group during phosphodiester bond cleavage, reminiscent of H47 in AcrIII-121. R112 from the neighbouring monomer likely provides a key electrostatic interaction for the cOA species during binding and catalysis.

Crn4 is functional in vivo
To explore a functional role of Crn4 in vivo, we reconstituted A. procaprae Crn4a into a well-developed recombinant type IIIA CRISPR system from Mycobacterium tuberculosis (MtbCsm), which produces a range of cOA species for effective activation of cOA-dependent effectors to provide plasmid immunity37. E. coli C43 cells expressing MtbCsm were transformed with a pRATDuet plasmid expressing an effector protein alone or along with Crn4a and carrying a tetracycline resistance gene (tetR). Fewer transformants were expected when the effector was activated by cOA generated by the MtbCsm system, which was programmed with a crRNA targeting a portion of tetR gene. As a control, we also tested MtbCsm programmed with a crRNA targeting the pUC plasmid, which is not activated to generate cOA in this experimental setup. Effectors tested in this programmed MtbCsm system were cA4-activated Csx1 from Thioalkalivibrio sulfidiphilus (TsuCsx1)27,38 and cA6-activated effectors Csm6 from M. tuberculosis (MtbCsm6)37 and A. procaprae Csm6-23.

In the absence of Crn4a, all three effectors were activated on transformation of the pRATDuet plasmid, providing effective resistance to plasmid transformation, reflected in reduced colony forming units (cfu) (Fig. 5 and Supplementary Fig. 11). The presence of Crn4a effectively alleviated plasmid immunity conferred by cOA-activated TsuCsx1 and MtbCsm6 (Fig. 5), suggesting that Crn4a reduces the concentration of both cA4 and cA6 when expressed in cells. In contrast, no significant difference in colony counts was observed for the Csm6-2 effector in the presence or absence of Crn4a. This may be because Csm6-2 is itself a very active ring nuclease; alternatively, this could reflect the relative affinities of cA6 binding by Crn4a and Csm6-2.

Methods
Cloning
The g-blocks of the crn4a and crn4b genes were codon optimised for expression in E. coli and purchased from Integrated DNA Technologies. Synthetic genes were cloned into the vector pEhisV5TEV8 between the NcoI and BamHI sites. The E. coli DH5a strain was used for cloning, and sequence integrity was confirmed by sequencing (Eurofins Genomics). Site-directed mutagenesis was performed using primers with the desired mutations. All synthetic genes and primers used in this study are listed in Supplementary Table 1. For the plasmid challenge assay, the g-block of the crn4a gene was cloned into the MCS-1 (Multiple Cloning Site-1) of pRATDuet37 between the NdeI and XhoI sites, under the control of a T7 promoter. For the expression of two proteins, the synthetic gene encoding TsuCsx137, MtbCsm637 or Csm6-2C3 was cloned into the MCS-2 (Multiple Cloning Site-2) of pRATDuet-Crn4 plasmid between the NcoI and HindIII sites, under the control of pBAD promoter.

Protein expression and purification
E. coli C43 (DE3) cells were transformed with plasmids for protein expression. 1 L of Luria-Broth (LB) containing cells transformed with the plasmid of interest was grown at 37°C to an OD600 of 0.6–0.8. Crn4a or Crn4b expression was induced with 0.4 mM isopropyl b-D-1-thiogalactopyranoside for 4 h at 25°C and 37°C, respectively. Cell pellets were harvested for purification by centrifugation at 4000 × g (Beckman Coulter Avanti JXN-26; JLA 8.1 rotor) at 4°C for 15 min. Pellets were lysed by sonicating for 6 min with 1 min rest intervals on ice, before pelleting cell debris by ultracentrifugation at 160,000 × g (Beckman Coulter Optima L-90K, 70 Ti rotor) at 4°C for 30 min. Supernatants were subsequently loaded onto a 5 mL HisTrap FF column (GE Healthcare) equilibrated with lysis buffer. After washing away unbound proteins with 20 column volumes of lysis buffer, a linear gradient elution was conducted with pump A (lysis buffer) and B (elution buffer: 50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 0.5 M imidazole, and 10% glycerol). Fractions containing recombinant protein were collected and concentrated for his-tag removal by overnight dialysis with TEV protease (1 mg per 10 mg protein) in size exclusion chromatography (SEC) buffer (20 mM Tris-HCl pH 8.0, 0.25 M NaCl, 1 mM DTT, and 10% glycerol). The TEV-cleaved protein was recovered by second immobilised metal affinity chromatography (IMAC) and further purified by SEC (S200 16/60 column, GE Healthcare) in SEC buffer under isocratic flow. The identity and purity of proteins were verified using SDS-PAGE and the pure protein was aliquoted and stored at −70°C.

Ring nuclease activity
To examine the ring nuclease activity, enzymes were incubated with 200-fold molar excess of cOA species in 20 mM Tris-HCl, pH 7.5, 250 mM NaCl and 1 mM EDTA at 37°C for the time indicated. For calculation of rate constants of wild type Crn4a/b and Csm6-2, 500 nM enzyme was incubated with 100 μM cA6, cA3 or cA4, at 37°C for the time points 1, 2, 3, 5, 10, 15, 30 and 60 min. Reaction samples were quenched by mixing with two equivalent volumes of cold methanol and vortexed for 30 s, before centrifugation at 4°C for 15 min. The supernatant was transferred to a new tube and vacuum dried, before resuspension in H2O for HPLC. Substrate and cleaved products were quantified from triplicate measurements and data were fitted using a one phase exponential model (Y=Ymax*(1-e-k*X)) using Prism (Version 10.2.2 (341)).

HPLC
HPLC analysis was conducted on a UltiMate 3000 UHPLC system (Thermo Fisher Scientific) equipped with an Accucore™ C18 column (Thermo Fisher Scientific 2.1 × 100 mm, particle size 2.6 μm) for the time-course samples and a C18 column (Kinetex EVO 2.1 × 50 mm, particle size 2.6 μm) for other analyses. The absorbance was monitored at 260 nm and the column temperature was set at 40°C. Solvent A was 20 mM ammonium acetate, pH 8.5 and B was methanol. The flow rate was set at 0.4 ml/min for the Accucore™ C18 column and 0.3 ml/min for the EVO C18 column. Gradient elution followed the same procedure: 0–0.5 min, 1% B; 0.5–6 min, 1–15% B; 6–7 min, 100% B.

Protein crystallisation
Crn4a H15A, at 11 mg mL−1, was incubated at room temperature for 30 min with 1.2 molar excess of cA6. Crn4a and Crn4b were used at concentrations of 13 and 15 mg mL−1 respectively. Immediately prior to crystallisation proteins were centrifuged at 14000 × g. Sitting drop vapour diffusion experiments were set up at the nanoliter scale using commercially available crystallisation screens and incubated at 293 K. Optimised Crn4a H15A with cA6 crystals were grown from 23.8% PEG 3350, 0.2 M magnesium chloride and 0.1 M Bis-Tris, pH 5.5. Apo Crn4a crystals were grown from 0.1 M sodium acetate, pH 4.5 and 22% v/v PEG Smear Broad (Molecular Dimensions). Crn4b crystals grew from 20% PEG 6000, 10% ethylene glycol and 0.1 M calcium chloride. Crystals were cryoprotected with 25% glycerol prior to harvesting and cryo-cooling in liquid nitrogen.

X-ray data collection, structure solution, and refinement
X-ray data were collected at a wavelength of 0.9537 Å, 100 K, on beamline I04 at the Diamond Light Source, to 2.34 Å (apo Crn4a), 1.44 Å (Crn4a H15A with cA6) and 1.09 Å (apo Crn4b) resolution. Data were automatically processed using Xia244. The structures were solved by phasing the data using PhaserMR45 in the CCP4 suite46, using a model generated by AlphaFold347, with initial B-factors modelled in Phenix48. Model refinement was achieved by iterative cycles of REFMAC549, where hydrogen atoms were added for refinement but not output to the pdb files, and manual model manipulation in COOT50. For the Crn4a H15A with cA6 data, electron density for cA6 was clearly visible in the maximum likelihood/σA weighted Fo-Fc electron density map at 3σ. The coordinates for cA6 were generated in ChemDraw (Perkin Elmer) and the library was generated using Acedrg51, before fitting the molecule in COOT. The quality of each structure was monitored throughout using Molprobity52. Data and refinement statistics are shown in Supplementary Table 2. The coordinates and raw data have been validated and deposited in the Protein Data Bank with deposition codes 9SMA for Crn4a, 9QS9 for Crn4a H15A in complex with cA6, and 9R7B for Crn4b.

Plasmid challenge assay
Plasmids from the programmed type III MtbCsm system have been described, including pCsm1-5 (containing Csm interference genes cas10, csm3, csm4 and csm5 from M. tuberculosis and csm2 from M. canettii)37, pCRISPR-TetR and pCRISPR-pUC (containing M. tuberculosis cas6 and a CRISPR array targeting a tetracycline-resistance gene or a pUC multiple cloning site, respectively)37 and pRATDuet-effectors (carrying tetracycline-resistance gene and M. tuberculosis (Mtb) csm6 or Thioalkalivibrio sulfidiphilus (Tsu) csx1)53.

E. coli C43 (DE3) cells carrying plasmids pCsm1-5 and pCRISPR-TetR or pCRISPR-pUC were transformed with 100 ng of pRATDuet derived plasmids containing different effectors with or without a Crn4 ring nuclease. After recovering at 37 °C for 2 h, a ten fold serial dilution of cells was applied onto LB agar supplemented with 100 μg/ml ampicillin and 50 μg/ml spectinomycin for determination of cell density of recipients, onto LB agar containing additional 12.5 μg/ml tetracycline for determination of transformation efficiency and LB agar with additional 0.2% (w/v) D-lactose and 0.2% (w/v) L-arabinose to determine the plasmid immunity mediated by cOA-dependent effectors. Plates were incubated at 37 °C overnight, before taking images of plates. Technical duplicates of two biological replicates were performed. Colonies were counted to calculate colony forming units (cfu/ml) and analysed using an unpaired t test to compare the difference between systems in the presence or absence of Crn4 using Prism (Version 10.2.2).

Bioinformatic analyses
To search for Crn4 homologues in prokaryotes, we downloaded the 38,742 complete prokaryotic genomes from NCBI on March 25th 2024. For phage genomes we used the Millard database of 28,114 genomes (May 2024 release)31 and for plasmids the PLSDB database containing 59,895 sequences32. Based on our initial identification of two main clades for Crn4 homologues, separate HMM databases were created for Crn4a and Crn4b. These profiles were integrated into a previously published Snakemake pipeline developed for finding type III CRISPR-Cas loci, effectors and ring nucleases in prokaryotic and in phage genomes3. In short, the pipeline annotates type III CRISPR-Cas loci and searches them for ring nucleases based on HMM profiles and protein length limitations.

To create the phylogenetic tree (Fig. 1), prokaryotic Crn4a and Crn4b amino acid sequences were concatenated into one file, and aligned using Muscle54. A phylogenetic tree based on the alignment was created with FastTree55 using -wag and -gamma arguments. Two Crn4 homologues (genomes GCF_002285795.1 and GCF_002863905.1) were removed from the dataset due to missing cyclase domains in the accompanying Cas10. The tree was plotted and annotated in RStudio v. XX with ggtree56. The accompanying genomic neighbourhood visualisation was created by merging h information from several annotation files output by the pipeline (CCTyper, cATyper, RN_typer) into the original NCBI general features files (.gff). The enhanced annotations were visualised using ggtree’s geom_facet wrapper for gggenes and centered around Crn4. Custom rules were used to bin the genes into the categories shown in Fig. 1. Crn4 fusions with known effector proteins were discovered by modifying the snakemake pipeline to search for Crn4 without protein length limitations.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Chi et al. report the discovery and characterization of a new CRISPR ring nuclease Crn4. Through\nbioinformatic analysis, authors found two subclasses of Crn4, Crn4a and Crn4b that associated with the Csm6-2/CorA and\nCan2 effectors, respectively. They showed that both Crn4a and Crn4b can cleave the cOA molecules, with Crn4a targeting\nall cA3, cA4 and cA6 while Crn4b targeting only cA3 and cA4. These properties make Crn4 family of ring nucleases unique\ndue to their broad specificity and targeting cA3. Authors presented two ultra-high-resolution crystal structures, that of Crn4b\nand that of Crn4a-H15A-cA6, revealing their unique fold and cA6-recognition mechanism. Three of the identified key\ninteraction residues were mutated on Crn4a and the resulting variants were tested for cOA cleavage activities. Finally,\nauthors performed a plasmid challenge assay to demonstrate the function of the Crn4 ring nuclease in cells. Overall, this\nmanuscript is clearly written in a structured manner. The majority of experimental results support authors’ conclusions and\ncontribute to our understanding of the CRISPR associated ring nucleases. However, several concerns should be addressed\nprior to publication.\nMajor concerns:\n1) Givent the broad specificity of Crn4a, it may be of interests to know if it also cleaves linear polyA or even other cyclic\nnucleotides, perhaps at elevated concentrations;\n2) The structural analysis did not provide a rationale for the difference in cOA cleavage specificity between Crn4a and Crn4b\nor between Crn4 and other ring nucleases;\n3) It would be helpful to clarify, either by structural argument or additional experiments, if the b-hairpin conformation is an\ninherent difference between Crn4a and Crn4b or due to cA6-binding to Crn4a.\n4) Along the same line, can new experiments support that the difference in b-hairpin contribute to or account for the observd\ndifference in cOA specificity between Crn4a and Crn4b?\n5) The 1.09 – 1.44 are likely among the highest resolutions of currently known cOA-interacting enzymes. However, authors\ndid not take advantage of the high quality structures in revealing previously unknown features. Authors did not show a single\nelectron density map, for instance, around any described structural elements (i.e, protein-protein or cA6 interfaces). Did\nauthors add hydrogens during refinement and observed clear hydrogen bonds responsible for specificity? Are there metal\nions observed, for instance, mediating binding of cA6? Are there any water molecules around the cleavage sites? Are the\ntwo scissile phosphate bonds in an in-line conformation (after modeling His15 in)?\n6) Given the high resolutions, authors should list amino acid rotamer outlier statistics in Table S2. It would be interesting to\nsee if there are any meaningful outliers observed at these resolutions.\n7) LC-MS analysis was mentioned in the method but no experimental data were shown.\nMinor comments\n1. Figure 4 (especially panels A-D) needs proper labeles.\n2. The manuscript can benefit from a supplementary figure showing structural comparison among the Crn/Csm6/Csx1\nproteins, especially their cOA-binding modes. The conserved residue among them should be compared.\n3. Add secondary structure elements to the aligned sequences of Crn4 in Supplementary Figure 1. Highlight the b-hairpin\nmediating dimerization in both enzymes.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "This manuscript by Chi et al. presents a comprehensive study on Crn4, a novel family of ring nucleases associated with type\nIII-D CRISPR systems, combining bioinformatics, biochemistry, structural biology, and in vivo functional assays. The\nidentification of Crn4 with a unique protein fold and broad cOA substrate specificity (including cA₃, cA₄, and cA₆) significantly\nadvances our understanding of type III CRISPR signaling and regulation. The work is rigorous and impactful, but several key\nrevisions are needed before it can be considered for publication in Nat Commun.\nMajor points:\n1.The structure of Crn4a presents as a homodimeric assembly and shows symmetric binding for cA₆, with four adenine\nbases in the plane of the ring, and the remaining two axial to ring. This symmetric binding mode provides a structural basis\nfor its recognition of cA₆ and cA₄, which both possess an axis of two-fold symmetry. Strikingly, cA₃, which lacks such two-fold\nsymmetry, is degraded efficiently by Crn4a to A₂>P and A₁>P, indicating asymmetric cleavage. Therefore, the structure of\ncA₃-bound Crn4a would be quite valuable to directly visualize how this asymmetric cleavage is achieved. If obtaining this\nstructure is not feasible, can the authors provide a detailed discussion to address this point?\n2.Page 6, 2nd paragraph: the authors drew a conclusion that Csm6-2 cleaves cOA using its CARF domain rather than\nHEPN domain, based on the result that EDTA did not inhibit cOA cleavage. However, as previously noted (Du et al. 2024,\n43, 304), EDTA does not effectively inhibit HEPN domain RNase activity of TtCsm6. I recommend using a Csm6-2 mutant\nlacking HEPN domain activity to specifically address which domain is responsible for its intrinsic ring nuclease activity on\ncA₆.\n3.Page 7, 1st paragraph: In the structural analysis of cA₆ binding , the authors identify several residues (Arg92, Glu16,\nSer91, Arg112, etc.) involved in cA₆ interaction. However, these residues are not indicated in the figures. Please explicitly\nlabel these key residues in Fig. 4C and 4D to visually link the text description to the structural context. Also please show the\nlabeling for residues in Fig. 4A-B\n4.To support the claim of specific cA₆ binding, please include a panel in Fig. 4 showing the 2Fₒ- Fc electron density map for\nthe bound cA₆ ligand.\n5.Please standardize to residue abbreviations (one-letter or three-letter) in all text, figure legends, and labels."}, "author_response": "Response to REVIEWER COMMENTS\nWe thank all the reviewers for their expert and thoughtful comments. We address\neach of these point by point below. We feel that the resultant revised manuscript is\ngreatly improved due to their input.\nAlong with all required textual changes we have:\n Obtained and described a new crystal structure of Crn4a in the apo form,\nallowing detailed analysis of the conformational changes on cA6 binding.\n Added further data showing that Crn4a can cleave cA5 and polyA RNA.\n Added further analysis of the structures as requested.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Chi et al. report the discovery and characterization of a new\nCRISPR ring nuclease Crn4. Through bioinformatic analysis, authors found two\nsubclasses of Crn4, Crn4a and Crn4b that associated with the Csm6-2/CorA and\nCan2 effectors, respectively. They showed that both Crn4a and Crn4b can cleave\nthe cOA molecules, with Crn4a targeting all cA3, cA4 and cA6 while Crn4b targeting\nonly cA3 and cA4. These properties make Crn4 family of ring nucleases unique due\nto their broad specificity and targeting cA3. Authors presented two ultra-high-\nresolution crystal structures, that of Crn4b and that of Crn4a-H15A-cA6, revealing\ntheir unique fold and cA6-recognition mechanism. Three of the identified key\ninteraction residues were mutated on Crn4a and the resulting variants were tested\nfor cOA cleavage activities. Finally, authors performed a plasmid challenge assay to\ndemonstrate the function of the Crn4 ring nuclease in cells. Overall, this manuscript\nis clearly written in a structured manner. The majority of experimental results support\nauthors’ conclusions and contribute to our understanding of the CRISPR associated\nring nucleases. However, several concerns should be addressed prior to publication.\nMajor concerns:\n1) Given the broad specificity of Crn4a, it may be of interests to know if it also\ncleaves linear polyA or even other cyclic nucleotides, perhaps at elevated\nconcentrations;\nAs suggested, we tested Crn4a against linear polyA. At high concentrations, we\nobserved cleavage of the substrate to generate a linear A3 product, suggesting that\nlinear polyA can serve as a substrate mimic of cOA species. We also tested for\ncleavage of cAAG, but observed no cleavage, suggesting a strict preference for\nadenine bases. Finally, we tested for cleavage of cA5, which is synthesised by some\ntype III CRISPR systems but which has no known role as an activator – this was\ncleaved efficiently by Crn4a. These new data are shown in Supplementary Figure 7\nand described in the text:\nGiven the broad specificity of Crn4a, we also tested its ability to degrade\nnoncanonical substrates (Supplementary Figure 7). Perhaps unsurprisingly, cA ,\n5\nwhich is not known to be an activator of any CRISPR effector, was degraded\nefficiently by Crn4a. The CBASS signalling molecule cAAG was not a substrate,\nsuggesting a strict specificity for adenine bases, and a 12 nt polyA RNA substrate\nwas cleaved by Crn4a three nucleotides from the 3’ end. This suggests RNA can fold\ninto the active site and be cleaved by the enzyme. Previously, it was demonstrated\nthat the CARF domain of the Csx1 ribonuclease can bind linear tetra-adenylate RNA\ntails which activated the HEPN domain34. The physiological relevance of such\nobservations is questionable, given the slow reaction kinetics.\n2) The structural analysis did not provide a rationale for the difference in cOA\ncleavage specificity between Crn4a and Crn4b or between Crn4 and other ring\nnucleases;\nA good understanding of the relationship between cOA specificity and structure for\nthe two ring nucleases would require structures with cA3 or cA4 bound in each.\nUnfortunately, although we attempted to obtain such complexes for both Crn4a and\nCrn4b, the data always showed no ligand bound. In addition, the large\nconformational change observed upon binding of cA suggests that it is residues in\n6\nthe ‘open’ form of the dimer that is likely to be important for defining specificity, rather\nthan those seen in the closed form. This point is now discussed in the text:\nDespite our best efforts, we were unable to crystallise obtain a ligand-bound complex\nwith Crn4b, or indeed Crn4a in complex with cA or cA . Given the considerable\n3 4\nmovement observed for Crn4a upon binding of cA , and our suggestion that\n6\nspecificity may largely be driven by interactions with the ‘open’ form of Crn4, direct\ncomparison of the factors influencing specificity for substrate for each protein was\nnot possible. Given the broad substrate specificity of the enzyme, this remains an\nimportant topic for future study.\n3) It would be helpful to clarify, either by structural argument or additional\nexperiments, if the b-hairpin conformation is an inherent difference between Crn4a\nand Crn4b or due to cA6-binding to Crn4a.\n4) Along the same line, can new experiments support that the difference in b-hairpin\ncontribute to or account for the observd difference in cOA specificity between Crn4a\nand Crn4b?\nTo address comments 3 and 4, we solved the crystal structure of apo Crn4a (PDB\n9SMA) allowing direct comparisons to be made with the ligand-bound structure.\nThese are best viewed in Supplemental movies 1 and 2.\nAddressing point 3: the beta-hairpin conformation is similar in apo Crn4a and Crn4b\nstructures, but moves upon binding of cOA to Crn4a. We have added the following to\nthe text:\nComparison of the apo and cA -bound structures of a Crn4a monomer reveals that\n6\nthe globular part overlaps perfectly, but the extended b-hairpin bends upwards at an\n“elbow” when cA is bound (Figure 4G). In dimeric Crn4a, this movement translates\n6\nto the protein encasing the cyclic oligoadenylate upon binding (Supplementary\nmovies 1 and 2). This conformational change means the loops of the b-hairpins\ncome together to sit above the central cavity where cA binds (Supplementary Figure\n6\n9F).\nAddressing point 4: both Crn4a and Crn4b cleave cA and cA , so the only difference\n3 4\nin specificity is Crn4a’s ability to cleave cA . Superimposition of the apo Crn4a and\nperfectly, suggesting from a catalytic machinery viewpoint there is sufficient space\nfor Crn4b to accommodate cA . Our current hypothesis is that the specificity is likely\n6\ndetermined by the ‘open’ form of the dimer before the cOA becomes enclosed in the\nbinding cavity. We have added text to this effect:\nGiven the observation of relatively few interactions between cA and Crn4a (see\n6\nabove, Supplementary Figure 4D), it is possible that the protein residues displayed in\nthe ‘open’ form of the dimer are more important for recognition and binding of cOA,\nand once the cOA has been enclosed in the central cavity of the ‘closed’ dimer there\nis less need for interactions to guide binding or specificity, and only those important\nfor catalysis, or for correct positioning of catalytic residues, are required.\n5) The 1.09 – 1.44 are likely among the highest resolutions of currently known cOA-\ninteracting enzymes. However, authors did not take advantage of the high quality\nstructures in revealing previously unknown features. Authors did not show a single\nelectron density map, for instance, around any described structural elements (i.e,\nprotein-protein or cA6 interfaces). Did authors add hydrogens during refinement and\nobserved clear hydrogen bonds responsible for specificity? Are there metal ions\nobserved, for instance, mediating binding of cA6? Are there any water molecules\naround the cleavage sites? Are the two scissile phosphate bonds in an in-line\nconformation (after modeling His15 in)?\nWe have added an electron density figure (Supplementary Figure 9A) showing the\ndifference map for cA .\nPDB file. A sentence has been added to this effect in the Methods section:\nModel refinement was achieved by iterative cycles of REFMAC5 49, where hydrogen\natoms were added for refinement but not output to the pdb files, and manual model\nmanipulation in COOT 50.\nHydrogen bond interactions are discussed in the text and are shown in new\nSupplementary Figures 9D and 9E. Relatively few interactions are seen between cA\n6\nand Crn4a, and often through main chain atoms, which led us to the hypothesis that\nthe specificity is largely determined by the ‘open’ form of the dimer and once cA is\n6\nenclosed fewer interactions are required other than to orientate the catalytic\nresidues. Additional text shown in point 4 above has been added to the manuscript\nto discuss this.\nThere are two magnesium ions coordinated to two of the phosphate groups in cA ,\n6\nwhich have been shown in new Supplementary Figure 9E. These magnesium ions\ninteract with three water molecules, which have also been included in the figure.\nFinally, for the question about in-line attack, we have measured all the in-line\nangles, which are in the range 100-100 °, far from the optimum for in-line attack.\nThe\nfollowing text has been added to the results for this:\nThe inline angles formed by O2’-P-O5’ are between 100-110 ° around the ring, far\nfrom the angle of ~160 ° that is optimal for in-line attack by the 2’-OH nucleophile 21.\nThis may reflect the fact that cA is not the optimal substrate for this enzyme;\n6\nadditionally, the H15A variant crystallised here may differ from the wild-type enzyme\nin this respect.\n6) Given the high resolutions, authors should list amino acid rotamer outlier statistics\nin Table S2. It would be interesting to see if there are any meaningful outliers\nobserved at these resolutions.\nRotamer outlier statistics have been added to Table S2. There was a single rotamer\noutlier in the structure of Crn4a in complex with cA , but the rotamer was not\n6\ndeemed to be meaningful in the context of the article.\n7) LC-MS analysis was mentioned in the method but no experimental data were\nshown.\nThe referee is correct, this method was not used in the manuscript and has been\nremoved in the revision.\nMinor comments\n1. Figure 4 (especially panels A-D) needs proper labeles.\nFigure 4 (and associated supplementary figures) have been revised extensively, in\nresponse to this and other suggestions.\n2. The manuscript can benefit from a supplementary figure showing structural\ncomparison among the Crn/Csm6/Csx1 proteins, especially their cOA-binding\nmodes. The conserved residue among them should be compared.\nWe have considered this suggestion, but as Crn4 is completely unrelated to the\nother known ring nucleases we feel this type of comparison might be better suited to\na review paper.\n3. Add secondary structure elements to the aligned sequences of Crn4 in\nSupplementary Figure 1. Highlight the b-hairpin mediating dimerization in both\nenzymes.\nWe have added a figure to the supplementary data showing these details, as\nrequested (see Supplementary Figure 8).\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers\nwho co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nReviewer's Comments\nThis manuscript by Chi et al. presents a comprehensive study on Crn4, a novel\nfamily of ring nucleases associated with type III-D CRISPR systems, combining\nbioinformatics, biochemistry, structural biology, and in vivo functional assays. The\nidentification of Crn4 with a unique protein fold and broad cOA substrate specificity\n(including cA₃ , cA₄ , and cA₆ ) significantly advances our understanding of type III\nCRISPR signaling and regulation. The work is rigorous and impactful, but several\nkey revisions are needed before it can be considered for publication in Nat\nCommun.\nMajor points:\n1.The structure of Crn4a presents as a homodimeric assembly and shows symmetric\nbinding for cA₆ , with four adenine bases in the plane of the ring, and the remaining\ntwo axial to ring. This symmetric binding mode provides a structural basis for its\nrecognition of cA₆ and cA₄ , which both possess an axis of two-fold symmetry.\nStrikingly, cA₃ , which lacks such two-fold symmetry, is degraded efficiently by Crn4a\nto A₂ >P and A₁ >P, indicating asymmetric cleavage. Therefore, the structure of\ncA₃ -bound Crn4a would be quite valuable to directly visualize how this asymmetric\ncleavage is achieved. If obtaining this structure is not feasible, can the authors\nprovide a detailed discussion to address this point?\nThis is a good point. A good understanding of the relationship between cOA\nspecificity and the structures of the two ring nucleases would require structures of\neach with cA3 (and possibly cA4) bound. Unfortunately, although we attempted to\nobtain such complexes, the data always showed no ligand was bound. This point is\nnow discussed in the text:\nIn Results:\nDespite our best efforts, we were unable to crystallise a ligand-bound complex with\nCrn4b, or indeed Crn4a in complex with cA or cA . Given the considerable\n3 4\nmovement observed for Crn4a upon binding of cA and our suggestion that\n6\nspecificity may largely be driven by interactions with the ‘open’ form of Crn4, direct\ncomparison of the factors influencing specificity for substrate for each protein was\nnot possible. Given the broad substrate specificity of the enzyme, this remains an\nimportant topic for future study.\nIn Discussion:\nThe observation that cA is degraded efficiently by Crn4 is particularly intriguing,\n3\ngiven that the enzyme has a dimeric organisation with two identical active sites and\ntwo-fold symmetry. While this is ideal for binding of 2-fold symmetric substrates such\nas cA and cA , it poses a challenge for binding and cleavage of cA , which has 3-\n4 6 3\nfold symmetry. An understanding of cA binding and cleavage at the molecular level\n3\nrequires a co-crystal structure, but unfortunately we were unsuccessful in obtaining\nthis structure. In its absence, we can speculate that cA engages part of the binding\n3\nsite and one of the two catalytic sites, leading to asymmetric cleavage.\n2.Page 6, 2nd paragraph: the authors drew a conclusion that Csm6-2 cleaves cOA\nusing its CARF domain rather than HEPN domain, based on the result that EDTA did\nnot inhibit cOA cleavage. However, as previously noted (Du et al. 2024, 43, 304),\nEDTA does not effectively inhibit HEPN domain RNase activity of TtCsm6. I\nrecommend using a Csm6-2 mutant lacking HEPN domain activity to specifically\naddress which domain is responsible for its intrinsic ring nuclease activity on cA₆ .\nThanks for pointing this out. As Csm6-2 is not the focus of this paper we have opted\nto revise the text to make clear that the observed ring nuclease activity of Csm6-2\ncould be due to its CARF domain, HEPN domain, or a combination of the two.\n3.Page 7, 1st paragraph: In the structural analysis of cA₆ binding , the authors\nidentify several residues (Arg92, Glu16, Ser91, Arg112, etc.) involved in cA₆\ninteraction. However, these residues are not indicated in the figures. Please explicitly\nlabel these key residues in Fig. 4C and 4D to visually link the text description to the\nstructural context. Also please show the labeling for residues in Fig. 4A-B\nResidues are now labelled in the figures, as requested. Additional figures showing\nthe interactions have been added as Supplementary Figures 9D and 9E.\n4.To support the claim of specific cA₆ binding, please include a panel in Fig. 4\nshowing the 2Fₒ- Fc electron density map for the bound cA₆ ligand.\nThis has been included as Supplementary Figure 9A.\n5.Please standardize to residue abbreviations (one-letter or three-letter) in all text,\nfigure legends, and labels.\nThis has been done as requested, using the one letter codes.\nReviewer 1:\nDid authors intend to use “surprisingly” instead of “unsurprisingly” in the sentence “Perhaps\nunsurprisingly, cA5, which is not known to be an activator of any CRISPR effector, was degraded\nefficiently by Crn4a.” ?\nResponse: We meant unsurprisingly, given Crn4 cleaves all other cOA species. We have now\nclarified this in the text.\nIt is better to add the requirement for high-concentrations of polyA in the sentence “12 nt polyA\nRNA substrate was cleaved by Crn4a three nucleotides from the 3’ end at an elevated\nconcentration.”\nResponse: We rephrased for clarity: “and a high concentration of Crn4 cleaved a 12 nt polyA\nRNA substrate three nucleotides from the 3’ end.”\nAuthors could note that “The fact that Crn4a, which lacks a HEPN domain, can cleave polyA\nsuggests its substrate plasticity.”\nResponse: We added a phrase on substrate plasticity to the discussion.\nFigure S7. Panel A, can authors clarify what the cleavage products of cA3 and cA5 likely are?\nThey do not seem to be aligned on the chromatogram.\nResponse: We re-ran these samples and have labelled them in figure S7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors added a new crystal structure of the apo Crn4a, allowing structural comparison. Authors also performed cleavage\nexperiments to compare substrate specificities. Additional, all minor, comments are included below.\nDid authors intend to use “surprisingly” instead of “unsurprisingly” in the sentence “Perhaps unsurprisingly, cA5, which is not\nknown to be an activator of any CRISPR effector, was degraded efficiently by Crn4a.” ?\nIt is better to add the requirement for high-concentrations of polyA in the sentence “12 nt polyA RNA substrate was cleaved\nby Crn4a three nucleotides from the 3’ end at an elevated concentration.”\nAuthors could note that “The fact that Crn4a, which lacks a HEPN domain, can cleave polyA suggests its substrate\nplasticity.”\nFigure S7. Panel A, can authors clarify what the cleavage products of cA3 and cA5 likely are? They do not seem to be\naligned on the chromatogram.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "All of my concerns have been adequately addressed in the revised manuscript. I have no further comments and recommend\nacceptance for publication."}, "author_response": ""}]

Decision letter