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 domain^1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction endonuclease-like domain^3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases^5,6, 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.

Extended Data Fig. 1.. cA_n-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 % identity between the genes that encode the Cas10 complex (with % similarity in parenthesis. (b-e) Card1 digestion of (b) M13 ssDNA, performed two times, (c) pUT7 dsDNA plasmid (30 minutes), performed two times, (d) ΦX174 supercoiled dsDNA (30 minutes), performed two times, or (e) ΦX174 linearized dsDNA (30 minutes), performed two times, in the presence or absence of cA₄, visualized by agarose gel electrophoresis. (f) Card1 digestion of ΦX174 ssDNA (60 minutes) in the presence of cA₄ and different divalent cations, visualized by agarose gel electrophoresis; performed two times. (g) Overview of Card1 cleavage sites across the ΦX174 genome based on the 5’ end mapping of DNA degradation products obtained after 2 hours of digestion, per 1 million reads. There appears to be preferential cleavage sites that may reflect lack of Card1 access to secondary structures formed within the ssDNA molecule. 26.7% of cuts occur at the 25 most frequent positions. (h) Same as (g) but after the analysis of M13 ssDNA degradation products obtained after 2 hours of digestion. 31.1% of cuts occur at the 25 most frequent positions. (i) Fragment size distribution of the ΦX174 degradation products after 2 hours of Card1 digestion. The average fragment length (163.6 nucleotides) is marked by the dotted line. (j) Same as (i) but analyzing M13 degradation products after 2 hours 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.. cA_n-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 minutes in buffers containing either no divalent cation, or either Mn²⁺, Mg²⁺, Ca²⁺, or Zn²⁺; performed two times. (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 non-specific RNase 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 nor G₅-C-G₅ could not be tested due to its resistance to cleavage by RNases. 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 Mn. This results in direct competition between the DNase and RNase activities of Card1 in each reaction. For each pair, one oligonucleotide is labelled with Cy3 and the other with Cy5 fluorescent groups, and the two panels display the same gel imaged through different filters. All reactions were quenched after 15 minutes; performed one time.

Extended Data Fig. 3.. Energetics of binding of cAs to dimeric Card1 and Electrostatic surface representation of Card1 and its cA_n-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) K_D values determined from ITC binding studies of selected Card1 mutants Y340A and M42A. (e-g) Electrostatic surface views of apo (panel e), cA₄-bound (panel f) and cA₆-bound (panel 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 cA_n-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 while 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 1.2 σ for (panel d) two orthogonal views of bound cA₄ and (panel e) the Mn in the catalytic pocket in the cA₄-dimeric Card1 complex. (f, g) Fitting of electron density contoured at 1.2 σ for the CARF pocket (panel f) and the catalytic pocket with bound Mn cation (panel 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 1.2 σ for two orthogonal views of bound cA₆ (panel h) and key residues in the catalytic pocket (panel i) in the cA₆-dimeric Card1 complex.

Extended Data Fig. 5.. Conformational changes between apo- and cA_n-bound states of dimeric Card1, comparison of the dimeric REase 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 (panel a) and cA₆-Card1 complex (panel b). (c) Two views of the structure of cA₄-bound Card1. The dimeric alignment of the REase domains of Card1 are shown in a black box. Note the position of one end of helical segments shown in a red box. (d, e) Structures of type II restriction enzymes EndoMS (PDB: 5GKE) (panel d) and PspGI (PDB: 3BM3) (panel e) bound to a single central mismatch containing dsDNA. The black boxed regions highlight their pair of REase domains. (f) dsDNA substrates containing mismatches used for cleavage assays in (panel g) and (panel 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 two times. (h) cA₄-activated Card1 digestion of dsDNA containing either single, double, triple or quadruple central C•C mismatches; performed two times.

Extended Data Fig. 6.. Intermolecular contacts and Mn 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 Mn 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 (panel f) and ribbon (panel g) representations in a model of the cA₆-dimeric Card1 complex.

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

(a) RNA-seq of staphylococci harbouring pTarget and pCRISPR-Cas10^HD, and +Card1. At 0 minutes, targeting is induced by the addition of aTc, and cells are harvested after 3 minutes. An equal amount of RNA from Listeria seeligeri was added to all samples prior to RNA purification to allow absolute comparison between timepoints. 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 minutes of Card1 activity. (b) Like (a), but in cells carrying a catalytically dead Card1 (dCard1). (c) A comparison between the log₁₀ read depth for all individual chromosomal genes between +Card1 cells and dCard1 cells, at 3 minutes. 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-Cas10^HD, with either +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 the +Card1 and +Csm6 cells both experienced growth arrest. (d) Growth of staphylococci carrying different pCRISPR(+Card1) taken from six escaper colonies obtained at the end of the experiment in Figure 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. are reported. (e) Agarose gel electrophoresis of plasmid DNA was extracted from escaper cells grown in (d), 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. (f) Growth of staphylococci carrying different pCRISPR variants expressing Cas10^HD, measured as OD₆₀₀ after the addition of aTc to induce the production of cA₄ by the Cas10^HD complex. Mean of three biological triplicates ± s.e.m. are reported. (g) Enumeration of colony-forming units (cfu) within staphylococcal cultures carrying different pCRISPR variants expressing Cas10^HD where cA₄ production was activated by the addition of aTc. At the indicated times after induction aliquots were removed and plated on solid media with or without aTc to count the remaining viable cells. Mean of three biological replicates ± s.e.m are reported. (h) Growth of staphylococci carrying different pCRISPR(+Card1, Cas10^HD) taken from five escaper colonies obtained in (g), measured as OD₆₀₀ after the addition of aTc to induce the production of cA₄ by the Cas10^HD complex. Mean of three biological triplicates ± s.e.m. are 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 anti-phage 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 ~15. Mean of three biological triplicates ± s.e.m. are 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 ~15. Mean of three biological triplicates ± s.e.m. are reported. (d) Same as in (c) but targeting the gp43 transcript, at an MOI ~2. Mean of three biological triplicates ± s.e.m. are reported. (e) Enumeration of plaque-forming units (pfu) within staphylococcal cultures carrying different pCRISPR variants after infection with Φ12γ3 at an MOI ~10. At the indicated times after infection aliquots were removed and plated on top agar media seeded with a susceptible strain. Mean of three biological replicates ± s.e.m are 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 ~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. are reported. (g) Same as in (f) but targeting the ORF27 transcript. In both (f) and (g), Cas10^HD cells with +Card1 do not lyse from infection (as it is the case for Δspc cells), indicating an incomplete phage life cycle. Mean of three biological triplicates ± s.e.m. are reported. In (f) and (g), the data representing Cas9 and Δspc is from the same experiment.

Extended Data Fig. 9.. Comparison of the cA bound structures of dimeric Csm6, dimeric Csx1, monomeric Cam1 and monomeric Cap4 with the emphasis on domain alignment, overall structure, and interactions between bound cA 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 (panel e) and cA₄-dimeric Csm6 complex (PDB: 6O6V) (panel f). The secondary structure (helices and sheets) is shown above the sequences. The CARF domains are highlighted by yellow. The images were generated by PHENIX.refine and modified.

Extended Data Fig. 10.. Docking model of ssDNA two possible strand directionalities 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 REase domains of cA₄-Card1 complex.

(a) We used the HDOCK program to position a B-form ssDNA with the sequence ApCpT1pG2pA3 with one (labeled strand directionality 1) of two possible strand directionalities in the catalytic pocket whereby the cleavable phosphate (in bold) was positioned relative to the pair of divalent cations coordinated to the catalytic acidic residues (see red arrow). One divalent cation (labeled 1) was observed in the x-ray structure of cA₄-bound Card1, while the other (labeled 2) was modeled based on its position in the structure of the NgoMIV restriction enzyme-DNA complex (PDB: 1FIU) ⁴⁹, 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 REase domain. It also shows positioning of the sugar-phosphate backbone flanking the cleavable phosphate (red arrow) of the modeled bound ssDNA within the REase 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, while 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 REase domain with an opposite directionality (labeled strand directionality 2). See the Methods section for modeling computations using strand directionality 2. (c) Side view of the modeled complex emphasizing the space available to readily accommodate ssDNAs (strand directionality 1) positioned in the pair of opposing REase pockets. (d) A top-down view of the modeled complex emphasizing the strand directionalities of the bound ssDNAs (strand directionality 1) positioned in the pair of opposing REase pockets.

Fig. 1.. Card1 is a cA₄-activated single-stranded DNase and RNase.

(a) Card1 cleavage of ΦX174 ssDNA for 30 minutes in the presence of cA₄ and cA₆; performed two times. (b) Cleavage preference of Card1, represented as a WebLogo, determined after next generation sequencing of ΦX174 degradation products. Five nucleotide positions upstream (−5 to −1) and downstream (1 to 5) of the detected cleavage sites are shown. (c) Card1 cleavage of a 60-nt ssRNA oligonucleotide for 15 minutes in the presence of cA₄ and cA₆; performed three times. (d) Crystal structure of dimeric apo-Card1 at 2.3 Å resolution. The monomers are colored yellow and magenta in the symmetrical dimer, with labeling of the CARF, hinge and REase domains, and highlighting the central hole between domains. (e) Crystal structure of co-crystallized cA₄-bound to dimeric Card1 at 3.0 Å resolution, with one cA₄ (in space-filling representation) bound per dimer and positioned within the periphery of the central hole. In addition, one Mn (in green) per monomer is bound in each REase catalytic pocket. (f) Conformational transitions following superposition of apo- (in silver) and cA₄-bound (in color) states of dimeric Card1.

Fig. 2.. Identification of Card1 residues essential for cA₄ activation and nucleic acid degradation.

(a) Superposition of one monomer module of apo- (in silver) and cA₄-bound (in color) Card1 states. (b) Highlight of the changes in loop segments in the vicinity of bound cA₄ (see arrows). (c) Highlight of the changes in the Card1 catalytic pocket with an emphasis on the side chain of E308 following Mn coordination on complex formation. (d, e) Intermolecular hydrogen bonds (d) and hydrophobic interactions (e) between bound cA₄ and amino acids of dimeric Card1. (f) Impact of mutations of the residues identified in (d, e) on in vitro ΦX174 ssDNA cleavage activity; performed three times. (g) Same as (f) but using a fluorescently labeled 60-nt ssRNA substrate; performed two times. (h) Lack of conformational transitions following superposition of apo- (in silver) and cA₆-bound (in color) states of dimeric Card1.

Fig. 3.. Card1 activation leads to a growth arrest of the cell and promotes destruction of target plasmids.

(a) Growth of staphylococci carrying pTarget and different pCRISPR variants, measured as OD₆₀₀ after the addition of aTc, in the absence of antibiotic selection for pTarget. Mean of three biological triplicates ± s.e.m. are reported. (b) Enumeration of colony-forming units (cfu) from staphylococcal cultures carrying different pCRISPR variants after the addition of aTc. At the indicated times after induction, aliquots were removed and plated on solid media with or without aTc to count the remaining viable cells. Mean of three biological replicates ± s.e.m are reported. (c-d) pTarget plasmid curing assay, where plasmid DNA was extracted from cells containing pTarget and different pCRISPR plasmids after addition of aTc. Plasmids were linearized and visualized by gel electrophoresis; performed three times.

Fig. 4.. Card1 protects staphylococci from phage infection.

(a) Growth of staphylococci carrying different pCRISPR variants programmed to target the ORF9 transcript of Φ12γ3, measured as OD₆₀₀ at different times after infection at a multiplicity of infection (MOI) between 2 and 8. Mean of three biological triplicates ± s.e.m. are reported. (b) Same as in (a) but targeting the ORF27 transcript at an MOI ~8. Mean of three biological triplicates ± s.e.m. are reported. (c) Same as in (b) but following cultures carrying different mutations in the cA₄ binding pocket of Card1, at an MOI ~15. (d) Enumeration of plaque-forming units (pfu) within staphylococcal cultures carrying different pCRISPR variants, at the indicated times after infection with Φ12γ3 at an MOI ~10. Mean of three biological replicates ± s.e.m are reported. Significant p values (p<0.05), obtained with two-sided t-test, are shown.