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The Card1 nuclease provides defence during type III CRISPR immunity

Abstract

In the type III CRISPR–Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded—but not double-stranded—nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.

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Fig. 1: Card1 is a cA4-activated ssDNase and ssRNase.
Fig. 2: Identification of Card1 residues that are essential for cA4 activation and nucleic acid degradation.
Fig. 3: Card1 activation leads to a growth arrest of the cell and promotes destruction of target plasmids.
Fig. 4: Card1 protects staphylococci from phage infection.

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Data availability

The atomic coordinates have been deposited in the PDB with the codes 6WXW (apo Card1), 6WXX (cA4–Card1 complex), 6WXY (cA6–Card1 complex) and 6XL1 (cA4–Card1(D294N) complex). The raw data for the DNA-sequencing and RNA-seq experiments performed in this study are found at the Sequence Read Archive (NIH) through accession code PRJNA672128.

Code availability

Custom Python code is available upon request to the corresponding authors.

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Acknowledgements

We thank the HTS core facility of the Rockefeller University for assistance with ITC experiments, and the Rockefeller University Genomics Resource Center for performing the Card1 in vivo ribonuclease NextSeq NGS experiment. J.T.R. was supported by a Boehringer Ingelheim Fonds PhD fellowship. L.A.M. is supported by a Burroughs Wellcome Fund PATH Award and a National Institutes of Health (NIH) Director’s Pioneer Award (DP1GM128184). L.A.M. is an investigator of the Howard Hughes Medical Institute. D.J.P. is supported by funds from the Geoffrey Beene Cancer Research Center, by NIH GM129430 and by a Memorial Sloan Kettering Cancer Center Core Grant (P30CA008748). This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US NIH (NIGMS P30 GM124165). The Pilatus 6M detector on 24‐ID‐C beamline is funded by a NIH‐ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE‐AC02‐06CH11357, and those of the Minnesota Supercomputing Institute.

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Authors and Affiliations

Authors

Contributions

J.T.R. and L.A.M. conceived the study. J.T.R. performed in vitro Card1 cleavage assays and NGS of its products, as well as all the in vivo experiments with the help of K.K. and R.F. W.X. performed Card1 biochemistry assays and all structural experiments. V.K. performed structural simulations and modelling. P.M. provided reagents and wrote a custom Python code. J.T.R., W.X., D.J.P. and L.A.M. wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Dinshaw J. Patel or Luciano A. Marraffini.

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Competing interests

L.A.M. is a cofounder and scientific advisory board member of Intellia Therapeutics and a cofounder of Eligo Biosciences. A US patent related to this work has been filed.

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Peer review information Nature thanks Philip Kranzusch, John van der Oost and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 cAn-mediated cleavage of ssDNA by Card1 at 37 °C.

a, Schematic of the S. epidermidis type III-A locus, showing the replacement of csm6 by card1 and the different mutations investigated in this study. A comparison with the T. succinifaciens type III-A locus is provided. Numbers indicate the per cent identity between the genes that encode the Cas10 complex (with per cent similarity in parenthesis). be, Card1 digestion of M13 ssDNA (performed twice) (b), pUT7 dsDNA plasmid (30 min) (performed twice) (c), ΦX174 supercoiled dsDNA (30 min) (performed twice) (d) or ΦX174 linearized dsDNA (30 min) (performed twice) (e), in the presence or absence of cA4, visualized by agarose gel electrophoresis. f, Card1 digestion of ΦX174 ssDNA (60 min) in the presence of cA4 and different divalent cations, visualized by agarose gel electrophoresis; performed twice. g, Overview of Card1 cleavage sites across the ΦX174 genome based on the 5′-end mapping of DNA degradation products obtained after 2 h of digestion, per 1 million reads. There appear to be preferential cleavage sites that may reflect lack of Card1 access to secondary structures formed within the ssDNA molecule. Of the cuts, 26.7% occur at the 25 most frequent positions. h, As in g, but after the analysis of M13 ssDNA degradation products obtained after 2 h of digestion. Of the cuts, 31.1% occur at the 25 most frequent positions. i, Fragment size distribution of the ΦX174 degradation products after 2 h of Card1 digestion. The average fragment length (163.6 nucleotides) is marked by the dotted line. j, As in i, but analysing M13 degradation products after 2 h of digestion. The average fragment length (150.1 nucleotides) is marked by the dotted line. k, Cleavage preference of Card1, represented as a WebLogo, determined after NGS of M13 degradation products. Five nucleotide positions upstream (−5 to −1) and downstream (1 to 5) of the detected cleavage sites are shown.

Extended Data Fig. 2 cAn-mediated cleavage of ssRNA by Card1 at 37 °C.

a, Card1 digestion of a ssRNA or a dsRNA molecular weight ladder. Card1 rapidly degrades the ssRNA, but not the dsRNA, ladder; performed three times. b, Digestion of a 60-nucleotide (nt) RNA species for 15 min in buffers containing either no divalent cation, or Mn2+, Mg2+, Ca2+ or Zn2+; performed twice. c, Cleavage of RNA oligonucleotides containing a fluorophore–quencher pair, measured as the increase in fluorescence, by Card1 with or without cA4, or with the nonspecific ribonuclease I as a positive control. The RNA oligonucleotides are either poly-A15, poly-C15 or poly-U15. Poly-G could not be synthesized, and cleavage of G5–A–G5 or G5–C–G5 could not be tested owing to their resistance to cleavage by ribonucleases. Each bar represents the mean of three replicates, ±s.e.m., given as relative fluorescent units. d, Simultaneous Card1 digestion of a pair of 30-nt DNA and RNA oligonucleotides, or of a pair of 50-nt DNA or RNA oligonucleotides, with increasing concentration of Card1 and cA4 in the presence of manganese. This results in direct competition between the deoxyribonuclease and ribonuclease activities of Card1 in each reaction. For each pair, one oligonucleotide is labelled with a Cy3 and the other with a Cy5 fluorescent group, and the two panels display the same gel imaged through different filters. All reactions were quenched after 15 min; performed once.

Extended Data Fig. 3 Energetics of binding of cyclic oligoadenylates to dimeric Card1 and electrostatic surface representation of Card1 and its cAn-bound complexes.

a, b, ITC curves for binding of cA4 (a) and cA6 (b) to dimeric Card1. c, No binding observed by ITC for cA4 binding to selected Card1 mutants (S11A, Y122A and I125A). d, Kd values determined from ITC binding studies of selected Card1 mutants (Y340A and M42A). eg, Electrostatic surface views of apo (e), cA4-bound (f) and cA6-bound (g) Card1. Electrostatic surface potentials were calculated in PyMol and contoured at ±75.

Extended Data Fig. 4 Oligomeric composition of the apo Card1 and cA4–Card1 complex in the crystal and in solution, and quality of 2Fo − Fc density maps in cA4- and cA6-bound structures of Card1.

a, SEC–MALS measurement of the molecular weight of apo Card1 in solution. The measured solution molecular weight of 88.1 kDa is close to the calculated molecular weight of the dimeric Card1 (90.0 kDa). b, Two alternate views of the head-to-tail dimer of dimers alignment of cA4–Card1 complex in the crystal. One dimer is shown in a ribbon and the other dimer is shown in a surface representation. c, SEC–MALS measurement of the molecular weight of cA4–Card1 complex in solution. The measured solution molecular weight of 87.6 kDa is close to the calculated molecular weight of the dimeric cA4–Card1 complex (91.2 kDa) rather than the tetrameric complex. d, e, Fitting of electron density contoured at 1.2σ for two orthogonal views of bound cA4 (d) and the manganese cation in the catalytic pocket in the cA4–dimeric Card1 complex (e). f, g, Fitting of electron density contoured at 1.2σ for the CARF pocket (f) and the catalytic pocket with bound manganese cation (g) (shown as a green ball) in the structure of the cA4–dimeric Card1(D294N) mutant complex. Bound waters (shown as red balls) can be observed in this 1.95 Å high-resolution structure of this mutant complex. h, i, Fitting of electron density contoured at 1.2σ for two orthogonal views of bound cA6 (h) and key residues in the catalytic pocket (i) in the cA6–dimeric Card1 complex.

Extended Data Fig. 5 Conformational changes between apo and cAn-bound states of dimeric Card1, comparison of the dimeric restriction endonuclease pockets in cA4-bound Card1 and type II restriction enzyme complexes with bound mismatch-containing dsDNA, and attempts at cA4-mediated cleavage of dsDNA containing central mismatches by Card1.

a, b, Vector lengths identify degree of conformational changes between dimeric apo Card1 and cA4–Card1 complex (a) or cA6–Card1 complex (b). c, Two views of the structure of cA4-bound Card1. The dimeric alignment of the restriction endonuclease domains of Card1 are shown in a black box. The position of one end of the helical segments is shown in a red box. d, e, Structures of type II restriction enzymes EndoMS (PDB 5GKE) (d) and PspGI (PDB 3BM3) (e) bound to a single central mismatch-containing dsDNA. The black boxed regions highlight their pair of restriction endonuclease domains. f, dsDNA substrates containing mismatches used for cleavage assays in g, h. g, cA4-activated Card1 digestion of dsDNA containing either no mismatch or a central C•C mismatch, as well as central mismatch-containing DNAs used in EndoMS (PDB 5GKE) and PfoI (PDB 6EKO) complexes. Cleavage of M13 ssDNA is shown as a control; performed twice. h, cA4-activated Card1 digestion of dsDNA containing either single, double, triple or quadruple central C•C mismatches; performed twice.

Extended Data Fig. 6 Intermolecular contacts and manganese coordination in the cA4–Card1 complex, comparative cleavage propensity of conversion of cA4 to ApA>p by Csm6 and Card1 in a time-dependent manner, and structure of the cA6-Card1 complex and clashes between cA6 and dimeric Card1 loop residues in a model of cA6 in the bound state.

a, Intermolecular hydrophobic interactions between bound cA4 and amino acids of dimeric Card1. b, Amino acids lining the catalytic pocket of the structure of the cA4–Card1(D294) complex. The bound manganese is shown as a green ball. The water molecules are shown as red balls. c, Mono-Q column analysis of cA4 cleavage by Csm6. d, Mono-Q column analysis of the time-dependent stability of cA4 incubating with Card1. e, Structure of the cA6–dimeric Card1 complex. f, g, Clash between cA6 and L339 of loop residues of Card1 in space-filling (f) and ribbon (g) representations in a model of the cA6–dimeric Card1 complex.

Extended Data Fig. 7 The ribonuclease activity of Card1 is not detected in vivo, although Card1 activation leads to a growth arrest.

a, RNA-seq of staphylococci containing pTarget and pCRISPR(Cas10HD and +Card1). At 0 min, targeting is induced by the addition of aTc, and cells are collected after 3 min. An equal amount of RNA from L. seeligeri was added to all samples before RNA purification to allow absolute comparison between time points. Each dot represents a gene, and is the average of two biological replicates. Genes that fall on or near the identity line are unchanged by 3 min of Card1 activity. b, As in a, but in cells carrying a catalytically dead Card1 (dCard1). c, A comparison between the log10-transformed read depth for all individual chromosomal genes between +Card1 cells and dCard1 cells, at 3 min. A value of 0 means that a gene showed no difference between +Card1 and dCard1 cells. Overall, there is no clear trend for depletion (or enrichment) in +Card1 cells relative to dCard1 cells. d, Northern blot analysis of cells carrying pTarget and pCRISPR(Cas10HD), with +Card1, dCard1, +Csm6 or dCsm6; performed three times. Targeting was induced at time 0 with the addition of aTc, and RNA was analysed with probes specific to the protospacer target transcript (in pTarget), the plasmid replication gene repF (in pTarget), the def gene (peptide deformylase, in the S. aureus chromosome) or the msaB gene (in the msaABCR operon, in the S. aureus chromosome). 5S rRNA is used as a loading control. Card1 activation showed no detectable RNA degradation, in contrast to robust RNA depletion following Csm6 activation. OD600 measurements confirmed that both the +Card1 and the +Csm6 cells experienced growth arrest. e, Growth of staphylococci carrying different pCRISPR(+Card1) taken from six escaper colonies obtained at the end of the experiment in Fig. 3c, measured as OD600 after the addition of aTc to induce the production of cA4 by the Cas10 complex. Mean of three biological triplicates, ±s.e.m., is reported. f, Agarose gel electrophoresis of plasmid DNA was extracted from escaper cells grown in e, showing deletions in pTarget or pCRISPR. Sanger sequencing determined the same promoter deletion in pTarget escapers 1–3, and similar pCRISPR deletions in escapers 4–6, all comprising the whole CRISPR-cas locus. g, Growth of staphylococci carrying different pCRISPR variants expressing Cas10HD, measured as OD600 after the addition of aTc to induce the production of cA4 by the Cas10HD complex. Mean of three biological triplicates, ±s.e.m., is reported. h, Enumeration of colony-forming units (cfu) within staphylococcal cultures carrying different pCRISPR variants expressing Cas10HD, in which cA4 production was activated by the addition of aTc. At the indicated times after induction, aliquots were removed and plated on solid medium with or without aTc to count the remaining viable cells. Mean of three biological replicates, ±s.e.m., is reported. i, Growth of staphylococci carrying different pCRISPR(+Card1, Cas10HD) taken from five escaper colonies obtained in g, measured as OD600 after the addition of aTc to induce the production of cA4 by the Cas10HD complex. Mean of three biological triplicates, ± s.e.m., is reported. j, Agarose gel electrophoresis of plasmid DNA was extracted from escaper cells grown in i, showing deletions in pTarget. Sanger sequencing determined the same deletion in pTarget escapers 1–5, comprising both the promoter and target sequences.

Extended Data Fig. 8 Card1-mediated antiphage immunity.

a, Schematic of the genomes of the staphylococcal phages used in this study (Φ12γ3 and ΦNM1γ6), showing the location of the transcripts targeted by the type III-A CRISPR–Cas system. Grey arrows indicate promoters. b, Growth of staphylococci carrying different pCRISPR variants with mutations in the catalytic pocket of Card1, programmed to target the ORF27 transcript of Φ12γ3, measured as OD600 at different times after infection, at an MOI of about 15. Mean of three biological triplicates, ±s.e.m., is reported. c, Growth of staphylococci carrying different pCRISPR variants programmed to target the gp14 transcript of ΦNM1γ6, measured as OD600 at different times after infection, at an MOI of about 15. Mean of three biological triplicates, ± s.e.m., is reported. d, As in c, but targeting the gp43 transcript, at an MOI of about 2. Mean of three biological triplicates, ±s.e.m., is reported. e, Enumeration of plaque-forming units (pfu) within staphylococcal cultures carrying different pCRISPR variants after infection with Φ12γ3 at an MOI of about 10. At the indicated times after infection, aliquots were removed and plated on top agar medium seeded with a susceptible strain. Mean of three biological replicates, ±s.e.m., is reported. f, Growth of staphylococci carrying different pCRISPR variants programmed to target the ORF9 transcript of Φ12γ3, measured as OD600 at different times after infection at an MOI of about 25. The immunity provided by the Cas9 nuclease, which directly recognizes and cleaves the phage genome shortly after its injection and therefore allows the survival of the infected cells, is used as a control to show that the observed growth delays are not due to an excessive amount of phage added in the experiment. Mean of three biological triplicates, ±s.e.m., is reported. g, As in f, but targeting the ORF27 transcript. In f, g, Cas10HD cells with +Card1 do not lyse from infection (as is the case for Δspc cells), indicating an incomplete phage life cycle. Mean of three biological triplicates, ±s.e.m., is reported. In f, g, the data representing Cas9 and Δspc are from the same experiment.

Extended Data Fig. 9 Comparison of the cyclic-oligoadenylate-bound structures of dimeric Csm6, dimeric Csx1, monomeric Cam1 and monomeric Cap4 with the emphasis on domain alignment, overall structure and interactions between bound cyclic oligoadenylate and catalytic residues in the dimeric CARF pockets and a comparative study of sequence and topology between cA4 complexes with dimeric Card1 and dimeric Csm6.

a, cA4–dimeric Csm6 complex (PDB 6O6V). b, cA4–dimeric Csx1 complex (PDB 6R9R). c, cA4–monomeric Can1 complex (PDB 6SCE). d, cA3–Cap4 monomeric complex (PDB 6VM6). There is a trimeric alignment of monomers in this complex. e, f, Sequence and topology of cA4–dimeric Card1 complex (e) and cA4–dimeric Csm6 complex (PDB 6O6V) (f). The secondary structure (helices and sheets) is shown above the sequences. The CARF domains are highlighted in yellow. The images were generated by phenix.refine and modified.

Extended Data Fig. 10 Docking model of two possible strand directionalities of ssDNA positioned in the catalytic pocket of one monomer of the structure of the cA4-bound Card1 complex and model of alignment of a pair of ssDNAs (strand directionality 1) within the catalytic pockets of the opposing restriction endonuclease domains of cA4–Card1 complex.

a, We used the HDOCK program33 to position a B-form ssDNA with the sequence ApCpT1pG2pA3 with one (labelled strand directionality 1) of two possible strand directionalities in the catalytic pocket, in which the cleavable phosphate (in bold) was positioned relative to the pair of divalent cations coordinated to the catalytic acidic residues (red arrow). One divalent cation (labelled 1) was observed in the X-ray structure of cA4-bound Card1, and the other (labelled 2) was modelled on the basis of its position in the structure of the NgoMIV restriction enzyme–DNA complex (PDB 1FIU)34, which exhibits a similar catalytic residue alignment. The model outlines hydrogen-bonding alignments (dashed lines) in the model of the T1–G2–T3 segment interacting with side chains of the restriction endonuclease domain. It also shows positioning of the sugar–phosphate backbone flanking the cleavable phosphate (red arrow) of the modelled bound ssDNA within the restriction endonuclease catalytic pocket, thereby outlining the alignment of the cleavable phosphate relative to the pair of divalent cations and acidic catalytic residues (E259 coordinated to cation E292 coordinated to cation 2, and D294 and E308 coordinated to both cations). A pair of lysine side chains (K310 and K328) form salt bridges (dashed lines) to the cleavage site and flanking phosphates. b, The ApCpT1pG2pT3 is bound to the restriction endonuclease domain with an opposite directionality (labelled strand directionality 2). Modelling computations using strand directionality 2 are provided in Methods. c, Side view of the modelled complex emphasizing the space available to readily accommodate ssDNAs (strand directionality 1) positioned in the pair of opposing restriction endonuclease pockets. d, A top-down view of the modelled complex emphasizing the strand directionalities of the bound ssDNAs (strand directionality 1) positioned in the pair of opposing restriction endonuclease pockets.

Supplementary information

Supplementary Tables

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Reporting Summary

Supplementary Figure 1

Raw gel images of Fig. 1c (a), Fig. 3d (b), Fig. 3c (c), Extended Data Fig. 1b (d), Extended Data Fig. 1d (e), Extended Data Fig. 1e (f), Extended Data Fig. 1f (g), Extended Data Fig. 2a (h), Extended Data Fig. 2b (i), Extended Data Fig. 2d (j-k), Extended Data Fig. 7d (l-p), Extended Data Fig.7f (q), Extended Data Fig. 7j (r), Fig. 1a (s), Fig. 2f (t), Fig. 2g (u-v), Extended Data Fig. 1c (w), Extended Data Fig. 5g (x), Extended Data Fig. 5h (y). For l-p, all images are mirrored, and p is a sample processing control.

Video 1

Conformational transitions spanning the entire dimeric topology on proceeding from apo-Card1 to cA4-Card1 D294N mutant and back. The Card1 dimer is shown in ribbon representation while the cA4 is show in space-filling representation. The Mn cations are shown as green balls and are observed in the structure of the complex.

Video 2

Conformational transitions spanning the cA4-binding pocket on proceeding from apo-Card1 to cA4-Card1 D294N mutant and back. On proceeding from apo-Card1 to its complex with bound cA4, Ile125 side chains rotate to sandwich A2 (and A4) between it and Pro 16, while side chains of Thr39 and Glu41 move together to recognize A1 (and A3) through two hydrogen bonds. See also Fig. 2a.

Video 3

Conformational transitions spanning the loop segments on proceeding from apo-Card1 to cA4-Card1 D294N mutant and back. On proceeding from apo-Card1 to its complex with bound cA4, side chains of Leu339 and Tyr340 move and rotate together to stack under bound cA4. See also Fig. 2c.

Video 4

Conformational transitions spanning the top half of the REase domains on proceeding from apo-Card1 to cA4-Card1 D294N mutant and back. On proceeding from apo-Card1 to its complex with bound cA4, the pair of hydrogen bonds between the side chains of Asn330 across the dimeric interface are broken, and replaced by hydrogen bonds between side chains of Asn330 and Ser335, as well as between side chains of Glu362 and Lys286/Lys336 across the dimeric interface.

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Rostøl, J.T., Xie, W., Kuryavyi, V. et al. The Card1 nuclease provides defence during type III CRISPR immunity. Nature 590, 624–629 (2021). https://doi.org/10.1038/s41586-021-03206-x

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