Materials and Methods

Cyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence

MATERIALS AND METHODSCloningEnzymes were purchased from Thermo Scientific, BioLine, Promega or New England Biolabs and used according to manufacturer's instructions. Oligonucleotides (Supplementary Table S2) and synthetic genes (Supplementary Table S3) were obtained from Integrated DNA Technologies (Coralville, IA, USA). Synthetic genes were codon-optimized for E. coli and restriction sites for cloning incorporated. All constructs were verified by sequencing (GATC Biotech, Eurofins Genomics, DE) unless stated otherwise. Protein concentrations were determined by UV quantitation (NanoDrop 2000, Thermo Scientific) using calculated extinction coefficients (ExPASy, ProtParam software) unless stated otherwise.CRISPR genes All genes were obtained as synthetic genes. To obtain N-terminal His6-fusion proteins, Mtb cas10 (csm1), cas11 (csm2), cas7 (csm3), cas5 (csm4), csm5 and csm6 were digested with NcoI and XhoI and ligated into the corresponding sites of the expression vector pEHisTEV (24). The Mtb cas6 gene was cloned into the NdeI and XhoI sites of the same vector to obtain the C-terminal His6-fusion. We cloned a truncated version of cas6, starting at Met53 relative to NCBI annotation, because the N-terminal region showed no homology to non-mycobacterial Cas6 proteins, and a transcription start site was identified within the predicted cas6 gene (25). Thioalkalivibrio sulfidiphilus csx1 was cloned into the NcoI and BamHI sites of pEV5HisTEV to obtain a N-terminal His8-fusion (13).CRISPR arrays A CRISPR array containing three Mtb repeat sequences flanking two identical spacer sequences targeting the pUC19 MCS was assembled by BamHI digest of the double-stranded mtbCRISPR array oligonucleotide (Supplementary Table S2) followed by ligation into pCDFDuet™-1 (Novagen, Merck Millipore). Mtb cas6 was cloned into MCS-2 to give pCRISPR.A CRISPR array consisting of four identical spacers targeting the tetracycline resistance gene, flanked by five Mtb repeat sequences, was assembled from phosphorylated oligonucleotides mtbCARep and mtbCA_TetR-Sp (Supplementary Table S2). The array was ligated into MCS-1 of pCDFDuet™-1 containing cas6 in MCS-2 to give pCRISPR-Target. Mtb Csm complex for in vitro studies Cas10 (csm1), csm2 and csm5 were cloned into pACYCDuet-1 (5′-NcoI, 3′-XhoI; Novagen, Merck Millipore), csm4 was cloned into pEHisTEV (5′-NcoI, 3′-XhoI) and csm3 was inserted into pEHisTEV (5′-NdeI, 3′-XhoI) by restriction enzyme cloning. These constructs were used as templates for assembly of M. tuberculosis CRISPR genes, including ribosome binding sites, into MultiColi™ (Geneva Biotech, Genève, CH) acceptor and donor vectors by sequence- and ligation-independent cloning (SLIC) as per manufacturer's instructions. Briefly, His6-csm4, csm2 and cas10 were introduced into the acceptor pACE; csm3, csm5 were cloned into pDK; Mca csm2 was inserted into pDC using restriction enzyme cloning (5′-NdeI, 3′-XhoI). The acceptor and donor plasmids were then recombined using Cre recombinase to give pCsm1–5. To obtain variant Csm complexes, His8-V5-csm4 (from pEHisV5TEV-Csm4), Mca csm2 (from pACYCDuet-Mca_Csm2) and cas10 (csm1) were introduced into pACE by SLIC; and csm3, csm5 were inserted into the donor plasmid pDK. Primer-directed mutagenesis was carried out on the assembled acceptor and donor plasmids, before Cre recombination to give pCsm1–5_C3 (Csm3 D35A) and pCsm1–5_Cy (Csm1 D630A/D631A). The identity of all donor and acceptor plasmids was verified by sequencing, and the final, recombined construct was analysed by restriction digest. Mtb Csm complex for in vivo studies The same templates as for construction of pCsm1–5_C3 were used with the addition of csm6 (pACYCDuet-1). The pACE assembly was as for pCsm1–5_C3. Csm3, csm5 and csm6 were introduced into pDK; for the ΔCsm6 plasmid, the donor plasmid en route to pCsm1–5 was used. Mutagenesis was carried out as before. All constructs were verified by sequencing. Suitable acceptor and donor plasmid combinations were then assembled by Cre recombination to give pCsm1–6, pCsm1–5ΔCsm6, pCsm1–6_C3, pCsm1–6_Cy and pCsm1–6_HD (Supplementary Table S1). The control plasmid pCsm_Control was obtained by Cre recombination of pACE and pDK without inserts. The identity of the final, recombined constructs was confirmed by restriction digest analysis.Construction of target plasmids An arabinose-inducible pRSFDuet™-1 (Novagen, Merck Millipore) derivative with tetracycline resistance was constructed as follows. The araC and pBAD region was polymerase chain reaction (PCR)-amplified from pBADHisTEV (Huanting Liu, University of St Andrews, UK) using primer pair ara-AvrII-R/F and cloned into the XbaI and NdeI sites of pRSFDuet™-1 to give pRSFara. The tetracycline resistance gene was PCR-amplified from the MultiColi™ vector pACE2 using the TetR primer pair; the pRSFara-backbone lacking its resistance gene was PCR-amplified using primer pair RSFara. The two PCR products were digested with SphI and BamHI and joined through ligation to give pRAT. The pUC19 MCS / lacZα gene was introduced into pRAT by megaprimer PCR (primer pair lacZ-pRAT) to give pRAT-Target (Supplementary Table S1).Protein production and purification Mtb Cas6 C-terminally His6-tagged Cas6 was expressed in BL21 (DE3) grown in LB containing 50 μg ml-1 kanamycin and 50 μg ml−1 chloramphenicol. An overnight culture was diluted 100-fold into fresh medium and grown at 37°C, 180 rpm to an OD600 of 0.4. The temperature was lowered to 16°C, and incubation continued until the OD600 reached 0.8. Production was induced with 50 μM IPTG, and the cultures were continued for a further 20 h. Cells were harvested by centrifugation. The pellet was resuspended in 50 mM Tris–HCl, 250 mM NaCl, 10 mM imidazole, 10% glycerol, pH 7.5, sonicated and the cell debris removed by centrifugation. The cleared lysate was loaded onto a pre-equilibrated HisTrap Crude FF (GE Healthcare) column, washed with 50 mM Tris–HCl, 250 mM NaCl, 30 mM imidazole, 10% glycerol, pH 7.5 and eluted in a gradient to 50 mM Tris, 250 mM NaCl, 500 mM imidazole, 10% glycerol, pH 7.5. Cas6-containing fractions were pooled and further purified by gel filtration (HiLoad 16/60 Superdex pg 200, GE Healthcare) in 50 mM Tris–HCl, 250 mM NaCl, 10% glycerol, pH 7.5. Cas6 and Cas6 H47N were concentrated as required using an Amicon Ultra centrifugal filter (MWCO 10 kDa, Merck-Millipore). Mtb Csm complex Escherichia coli C43(DE3) cells were co-transformed with the MultiColi™ construct pCsm1–5 (or pCsm1–5_C3, pCsm1–5_Cy) and pCRISPR. Overnight cultures were diluted 100-fold into LB containing 100 μg ml−1 ampicillin and 50 μg ml−1 spectinomycin, incubated at 37°C, 180 rpm until the OD600 reached 0.8. After induction with 100 μM IPTG, incubation was continued at 25°C for 5 h. Cells were harvested by centrifugation and pellets stored at −20°C. Cells were resuspended in lysis buffer (50 mM Tris–HCl, 500 mM NaCl, 10 mM imidazole, 10% glycerol, pH 7.5) and lysed by sonication. The cleared lysate was loaded onto HisPur Ni-NTA resin (Thermo Scientific), washed with lysis buffer and eluted with lysis buffer containing 250 mM imidazole. Csm complex-containing fractions were diluted 2-fold with lysis buffer to prevent aggregation, pooled, then dialysed at 4°C overnight in the presence of 0.3 mg ml−1 TEV protease against lysis buffer. The protein solution was passed through His-Pur Ni-NTA resin a second time, and the flow-through was concentrated using an Amicon Ultracentrifugal filter (30 kDa MWCO, Merck-Millipore) and further purified by gel filtration (HiPrep 16/60 Sephacryl pg 300 HR, GE Healthcare) using 20 mM Tris–HCl, 250 mM NaCl, 10% glycerol, pH 7.5 as mobile phase. When necessary, the Csm complex was further purified on MonoS 4.6/100 PE (GE Healthcare) in 20 mM MES, 50 mM NaCl, pH 6 and eluted using a salt gradient to 1 M NaCl. Single-use aliquots were flash-frozen and stored at −80°C. Mtb Csm6 and T. sulfidiphilus Csx1 Mtb Csm6 and TsuCsx1 were expressed in C43 cells. Cells were grown for 4 h at 25°C before harvest by centrifugation at 4000 rpm at 4°C for 15 min (JLA 8.1 rotor). Cells were lysed in buffer containing 50 mM Tris–HCl pH 8.0, 0.5 M NaCl, 10 mM imidazole and 10% glycerol supplemented with 1 mg/ml chicken egg lysozyme (Sigma-Aldrich) and one ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablet (Roche) by sonicating six times for 1 min with 1 min rest intervals on ice. Cell lysate was then ultracentrifugated at 40000 rpm (70 Ti rotor) at 4°C for 45 min and loaded onto a 5 ml HisTrap FF Crude column equilibrated with wash buffer containing 50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 30 mM imidazole and 10% glycerol. After washing unbound protein with 20 CV wash buffer, recombinant Csm6 or TsuCsx1 was eluted with a linear gradient of wash buffer supplemented with 0.5 M imidazole across 16 CV, holding at 10% for 2 CV, 20% for 4 CV and 50% for 3 CV. Csm6 could not be concentrated and was therefore further purified using MonoQ (GE Healthcare) anion exchange chromatography, diluting protein from nickel affinity fractions directly in buffer containing 20 mM Tris-HCl pH 7.5 and 50 mM NaCl and eluting protein across a linear gradient of buffer containing 20 mM Tris-HCl pH 7.5 and 1M NaCl. For TsuCsx1, nickel affinity fractions containing the protein were concentrated using a 10 kDa MWCO ultracentrifugal concentrator, and further purified by size exclusion chromatography (S200 26/60; GE Healthcare) in buffer containing 20 mM Tris-HCl pH 8.0, 0.5 M NaCl and 1 mM DTT. All proteins were aliquoted, flash frozen with liquid nitrogen, and stored at −80°C.AssaysCyclic oligonucleotides cA4 and cA6 were purchased from BIOLOG Life Science Institute (Bremen, DE).Oligonucleotides All RNA and DNA oligonucleotides as well as 6-FAM™-labelled RNA substrates were purchased from Integrated DNA Technologies (Leuven, BE). For nuclease assays, labelled oligonucleotides were gel purified as described previously (26). Duplexed DNA oligonucleotides were prepared by mixing equimolar amounts of ssDNA in 10 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, pH 8.0, heating to 95°C for 10 min, followed by slow cooling. Oligonucleotides were 5′-end-labelled with γ-32P-ATP (10 mCi ml−1, 3000 Ci mmol−1; Perkin Elmer) using polynucleotide kinase (Thermo Scientific). Oligonucleotides were quantified spectophotometrically using calculated extinction coefficients (OligoAnalyzer tool, IDT). RNA ladders were obtained by alkaline hydrolysis (Thermo Fisher Scientific, RNA Protocols).Cas6 ribonuclease assay The reaction mixture contained 3 μM Cas6 in 20 mM Tris–HCl, 100 mM potassium glutamate, 1 mM DTT, 5 mM EDTA, 0.1 U μl−1 SUPERase•In™ (Thermo Scientific), pH 7.5. Reactions were initiated by addition of 5′ 6-FAM™-labelled mtbRepeat_Cas6 RNA (Supplementary Table S2) to 50 nM final concentration. The samples were incubated for up to 1 h at 37 °C. The reaction was stopped by extraction with phenol-chloroform-isoamyl alcohol. RNA species were resolved on a 20% denaturing (7 M urea) acrylamide gel in 1X TBE buffer, and visualized by scanning (Typhoon FLA 7000, GE Healthcare).Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM 5′-32P-labelled target RNA (Supplementary Table S2) to 0.8 μM Csm, 5 mM MgCl2, 0.1 U μl−1 SUPERase•In™ (Thermo Scientific), 50 mM Tris–HCl, 50 mM NaCl, pH 8.0, and incubating at 30°C for up to 2 h. The reaction was stopped by phenol–chloroform–isoamyl alcohol extraction to remove proteins. P1.A26 RNA (Supplementary Table S2) was used as non-target RNA control. Products were separated by gel electrophoresis (20% denaturing polyacrylamide) and visualized by exposure to a BAS Storage Phosphor Screen (GE Healthcare).DNase activity of the Csm complex To test for DNase activity of the Csm complex, 25 nM 5′-32P-labelled DNA substrate was added to 0.8 μM Csm, 200 nM target RNA, 0.1 mM CoCl2, 0.1 U μl−1 SUPERase•In™ (Thermo Scientific), 50 mM Tris–HCl, 50 mM NaCl, pH 8.0. The solution was incubated at 30°C for 90 min, and the reaction stopped by phenol–chloroform extraction. Products were analysed as described above.Cyclic oligoadenylate production and LC-MS analysis Cyclic oligoadenylate production was triggered by adding 2 μM crude target RNA to 0.8 μM Csm, 1 mM adenosine triphosphate (ATP), 5 mM MgCl2, 0.1 U μl−1 SUPERase•In™ (Thermo Scientific), 50 mM Tris–HCl, 50 mM NaCl, pH 8.0, and incubating at 30°C for up to 2 h. The reaction was stoped by phenol–chloroform–isoamyl alcohol extraction. Reaction mixtures that were used for Csm6 or Tsu Csx1 activation assays were further extracted with chloroform–isoamyl alcohol to remove residual phenol. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system. Samples were desalted on C18 cartridges (Harvard Apparatus). Compounds were separated on a Kinetex 2.6 μm EVO C18 column (2.1 × 5 mm, Phenomenex) using the following gradient of acetonitrile (B) against 20 mM ammonium bicarbonate (A): 0–2 min 2% B, 2–10 min 2–8% B, 10–11 min 8–95% B, 11–14 min 95% B, 14–15 min 95–2% B, 15–20 min 2% B at a flow rate of 350 μl min−1 and column temperature of 40°C. UV data were recorded at 254 nm. Mass data were acquired on the FT mass analyser in positive ion mode with scan range m/z 200–2000 at a resolution of 30 000.Csm6 activation assay Csm6 (125 nM dimer) was incubated with 25 nM 5′-radiolabelled P1.A26 RNA substrate together with 2 μM cold RNA in 50 mM MES, 100 mM potassium l-glutamate, pH 6.5. The reaction was started by addition of synthetic cA6 or 0.1 volumes protein-free reaction mixture from the cyclic oligoadenylate production above. The reaction was stopped after 45 min at 37°C by phenol–chloroform extraction. Products were analysed by 20% denaturing polyacrylamide gel electrophoresis.TsuCsx1 activation assay Csx1 (500 nM dimer) was incubated with 50 nM 5′-radiolabelled A1 RNA substrate and 1 μM cA4, 1 μM cA6 or 1 μl Csm-derived cOA (see above) in pH 7.5 buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl and 1 mM DTT in a 10 μl reaction volume at 35°C. Reactions were stopped at 1, 5, 15 or 30 min by the addition of a reaction volume equivalent of phenol–chloroform and extracted products were analysed by denaturing gel electrophoresis as described above.Plasmid immunity in vivo pCsm1–6 and pCRISPR were co-transformed into E. coli C43(DE3). Plasmids were maintained by selection with 100 μg ml−1 ampicillin and 50 g ml−1 spectinomycin. Competent cells were prepared by diluting an overnight culture 50-fold into fresh, selective LB medium; the culture was incubated at 37°C, 220 rpm until the OD600 reached 0.8–1.0. Cells were collected by centrifugation and the pellet resuspended in an equal volume of 60 mM CaCl2, 25 mM MES, pH 5.8, 5 mM MgCl2, 5 mM MnCl2. Following incubation on ice for 1 h, cells were collected and resuspended in 0.1 volumes of the same buffer containing 10% glycerol. Aliquots (100 μl) were stored at −80°C. Target or control plasmid (100 ng pRAT-Target, 100 ng pRAT, respectively) were added to the competent cells, incubated on ice for 30 min and transformed by heat shock. LB medium (0.5 ml) was added and the transformation mixture incubated with shaking for 2.5 h before collecting the cells and resuspending in 100 μl LB. A total of 5 μl of a 10-fold serial dilution were applied in duplicate to LB agar plates supplemented with 100 μg ml−1 ampicillin and 50 g ml−1 spectinomycin when selecting for recipients only; transformants were selected on LB agar containing 100 μg ml−1 ampicillin, 50 g ml−1 spectinomycin, 25 μg ml−1 tetracycline, 0.2% (w/v) β-lactose and 0.2% (w/v) l-arabinose. Arabinose was omitted when transcriptional induction of target was not required. Plates were incubated at 37°C for 16–18 h. Colonies were counted manually and corrected for dilution and volume to obtain colony-forming units (cfu) ml−1, statistical analysis was performed with RStudio (RStudio Inc., Boston, USA, version 1.2.1335). The experiment was performed as two independent experiments with two biological replicates and at least two technical replicates.

Article TitleCyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence


The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogenMycobacterium tuberculosis. Here, we report a comprehensive analysis of thein vitroandin vivoactivities of the type III-AM. tuberculosisCRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate thatM.tuberculosishas a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

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