Materials and Methods

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence

MATERIALS AND METHODSCloningFor cloning, synthetic genes (g-blocks) encoding SthCan2, TsuCan2 and VC1899 (full sequences are shown in Supplementary Table S1), codon optimised for expression in Escherichia coli, were purchased from Integrated DNA Technologies (IDT), Coralville, USA and cloned into the pEHisV5TEV vector between the NcoI and BamHI sites (29). Competent DH5α (E. coli) cells were transformed with the construct and sequence integrity was confirmed by sequencing (GATC Biotech, Eurofins Genomics, DE). The plasmids were then transformed into E. coli C43 (DE3) cells for protein expression. The inactivated nuclease domain variants, E276A/D278A for SthCan2, E302A/K304A for TsuCan2 and E291A/D293A for VC1899 were expressed from plasmids where the QuikChange Site-Directed Mutagenesis kit was used to introduce mutations in the wild type genes as per manufacturer's instructions (Agilent Technologies; primers used for mutagenesis are shown in Supplementary Table S2).Protein production and purificationFor protein expression, the cells expressing each Can2 orthologue in LB medium were grown at 37°C to an OD600 of ∼0.8, and then expression was induced with 0.4 mM isopropyl-β-d-1-thiogalactoside (IPTG) and grown overnight at 25°C. Cells were harvested by centrifugation at 3063 × g at 4°C for 15 min and resuspended in buffer containing 50 mM Tris–HCl pH 7.5, 500 mM NaCl, 10 mM imidazole and 10% glycerol. A protease inhibitor tablet (Roche; one tablet per 100 ml) and lysozyme (Sigma-Aldrich; 1 mg/ml) were added to the cell suspension, and cells were lysed by sonicating six times for 1 min on ice with 1 min rest intervals. The lysate was cleared at 117 734 × g at 4°C for 45 min and loaded onto a pre-equilibrated 5 ml HisTrap FF crude column (GE Healthcare), washed with 5 column volumes (CV) of wash buffer containing 50 mM Tris–HCl pH 7.5, 500 mM NaCl, 30 mM imidazole and 10% glycerol and eluted with a step gradient (holding at 20% for 4 CV and 50% for 4 CV) of elution buffer containing 50 mM Tris–HCl pH 7.5, 500 mM NaCl, 500 mM imidazole and 10% glycerol. Can2-containing fractions were pooled and concentrated using a 10 kDa molecular mass cut-off centrifugal concentrator (Merck). Tobacco Etch Virus (TEV) protease (1 mg per 10 mg protein) was used to remove the polyhistidine affinity tag while dialysing in wash buffer overnight at room temperature. The protein was isolated from TEV protease by the HisTrap FF crude column. The unbound fraction was collected and buffer-exchanged into a buffer containing 50 mM MES pH 6.5 and 150 mM NaCl using a centrifugal concentrator. Can2 was further purified by size exclusion chromatography (S200 26/60; GE Healthcare) in buffer containing 50 mM MES pH 6.5 and 150 mM NaCl. After concentration, Can2 was aliquoted and frozen at –80°C. The three Can2 homologues, SthCan2, TsuCan2 and VC1899 were purified using the same method. Nuclease variants were purified by the same method as for the respective wild-type proteins.For seleno-methionine labelled expression of SthCan2, the plasmid containing the can2 gene was transformed into E. coli B834 (DE3) cells. Cells were grown in M9 minimal medium supplemented with Selenomethionine Nutrient Mix (Molecular Dimensions, Newmarket, Suffolk, UK) and 50 mg l−1 (l)-selenomethionine (Acros Organics). The protein was purified by the same method described for native SthCan2.Plasmid cleavage assays1.8 nM supercoiled pEV5HisTEV plasmid was incubated with SthCan2 (500 nM dimer; equivalent to 1 μM total protein as measured by absorbance at 280 nm) and its nuclease domain variant E276A/D278A (500 nM dimer) for the time indicated in Figure ​Figure2.2. Reactions were carried out at 50°C in 20 μl final reaction volume at pH 7.0 with the buffer containing 20 mM HEPES, 100 mM NaCl and 1 mM EDTA supplemented with 1 μM cA4 and 5 mM MnCl2. The standard migration positions for supercoiled, linear or open circle plasmid were ascertained from the incubation with buffer only, with BamHI (Thermo Scientific) or with nicking endonuclease Nt.BspQI (New England BioLabs), respectively. Control reactions included incubating plasmid without protein, MnCl2 or cA4. For single-turnover kinetics experiments, triplicate experiments were carried by incubating SthCan2 (500 nM dimer) with plasmid substrate (1.8 nM) under the same conditions and stopped at the indicated times by adding 10 mM EDTA. All reactions were analysed by 0.7% agarose gel electrophoresis. Gels were scanned and quantified as described previously (19). Nicked and linearized plasmids are considered as products. This value is divided by the total of products plus substrates, to give the fraction cleaved. The data were plotted against time using Kaleidagraph (Synergy Software) and fitted to a single exponential curve as previously described (19).Open in a separate windowFigure 2.Can2 is activated by cA4 to degrade scDNA and RNA. (A) Agarose gel analysis of supercoiled plasmid (1.8 nM) nicking and degrading activity by SthCan2 and TsuCan2 (500 nM dimer). Supercoiled plasmid was degraded rapidly by SthCan2 in the presence of cA4 (1 μM) and MnCl2 (5 mM). Plasmid was incubated with wild-type SthCan2 at 50°C for 0.5, 1, 2 and 5 min in reaction buffer supplemented with cA4 and MnCl2. The nuclease variant E276A/D278A was incubated under the same conditions for 30 min. Wild-type TsuCan2 and its nuclease variant E302A/K304A were incubated under the same conditions for 30 min at 37 or 50°C. Standards corresponding to supercoiled (SC), linear and nicked plasmid are shown after the marker (M) lane. Control lanes C1, C2 and C3 show the reactions incubated for 30 min without protein, MnCl2 and cA4, respectively. (B) Single-turnover kinetic analysis of scDNA cleavage by SthCan2 and Can1 (the plot for Can1 is from our previous study (19)). SthCan2 (500 nM dimer) was incubated with scDNA (1.8 nM) under the same conditions as in part A and the reaction was stopped at 10 s, 20 s, 40 s, 1 min, 2 min and 3 min. The cleaved fraction of scDNA was plotted against time and fitted to a single exponential curve as described in Materials and Methods. The rate constant of scDNA cleavage for SthCan2 and Can1 are 3.39 ± 0.57 and 0.81 ± 0.15 min−1, respectively. Values and error bars represent the mean of triplicate experiments and the standard deviation. (C) Plot of fluorescent signals emitted by RNaseAlert substrates when they were cleaved by wild-type SthCan2, TsuCan2 or VC1899. RNaseAlert substrates (30 nM) were incubated with the enzymes (500 nM dimer) in reaction buffer and supplemented with cA4 (1 μM) and MnCl2 (5 mM) at 37°C. The fluorescent signal was plotted against time. Values and error bars represent the mean of triplicate experiments and the standard deviation.To test DNase activity of proteins VC1899 or TsuCan2, 1.8 nM plasmid was incubated with 500 nM dimer protein at 37°C or 50°C in 20 μl final volume in the same buffer as described above. Reactions were supplemented with 1 μM cA4 and 5 mM MnCl2 as indicated in Supplementary Figure S1. All reactions were stopped by adding 10 mM EDTA and analysed by 0.7% agarose gel electrophoresis.RNA cleavage assays5′-FAM labelled ssRNA (30 nM) was incubated with SthCan2 (500 nM dimer) and E276A/D278A (500 nM dimer) for the desired time. The sequence of the ssRNA is listed in Supplementary Table S2. Reactions were carried out at 50°C in 20 μl final reaction volume in pH 7.0 buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM EDTA and three units SUPERase•In Inhibitor (Thermo Scientific) supplemented with 1 μM cA4 and 5 mM MnCl2 or MgCl2. Control reactions included incubating RNA in buffer only, and incubating RNA without protein, metal ion or cA4. Experiments were carried out in triplicate for single-turnover kinetics and quenched by adding to five reaction volumes of phenol chloroform (Ambion) and vortexing. All reactions were analysed by 20% denaturing PAGE (20% acrylamide, 7 M urea and 1× Tris/borate/EDTA (TBE)). Gels were scanned and quantified as described previously (19). The uncleaved ssRNA at each time point was divided by the negative control to give the fraction uncleaved. The fraction cleaved was plotted against the time using Kaleidagraph (Synergy Software) and fitted to a single exponential curve as previously described (19). To test RNase activity of homologous proteins VC1899 or TsuCan1, reactions were carried out at 37°C in the same conditions.RNaseAlert fluorimetric assayRNaseAlert substrates were purchased from Integrated DNA Technologies (IDT). 30 nM substrate was incubated with different enzymes (500 nM dimer) at 37°C for 35 min. Reactions were carried out in a 30 μl final reaction volume in a pH 7.0 buffer containing 20 mM HEPES, 100 mM NaCl, 1 mM EDTA and three units SUPERase•In Inhibitor (Thermo Scientific), supplemented with 1 μM cA4 and 5 mM MnCl2. The substrates are fluorescence-quenched oligonucleotide probes that emit fluorescence signal after being cleaved. The signal was detected using a microplate reader (FLUOstar Omega, BMG LABTECH) with excitation and emission wavelengths set at 485 and 520 nm, respectively.Radiolabelled cA4 cleavage assays 32P labelled cA4 was generated by incubating S. solfataricus type III-D complex with α-32P-ATP as described previously (30). For cA4 cleavage assays, ∼10 nM radiolabelled cA4 was incubated with different concentrations of SthCan2. Reactions were carried out at 50°C for 30 min in 20 μl final volume in 20 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0 and three units SUPERase•In Inhibitor supplemented with 5 mM MgCl2. The control reaction was carried out by incubating radiolabelled cA4 in buffer only under the same conditions. All reactions were quenched and deproteinized by phenol-chloroform extraction, then chloroform extraction, before loading onto thin-layer chromatography (TLC) plates. Plates were visualized by phosphor imaging.Plasmid ligation and supercoiling1.5 nM pEV5HisTEV plasmid was incubated with SthCan2 (100 nM dimer) at 50°C for 1.5 min in the pH 7.0 buffer described above, supplemented with 200 μM cA4 and 5 mM MnCl2. Reactions were quenched and deproteinized by PCR Clean-Up System (Promega). The eluted product was incubated with DNA ligase and gyrase as described in a previous study (19). Nicking endonuclease Nt.BspQI was used as a positive control. All reactions were analysed by 0.7% agarose gel electrophoresis.Plasmid transformation assayThe construction of the pCsm1-5ΔCsm6 plasmid (containing the type III Csm interference genes cas10 (csm1), csm3, csm4, csm5 from M. tuberculosis and csm2 from M. canettii) and pCsm1-5_Cy plasmid expressing an inactivated cyclase variant (Csm1 D630A/D631A) of the Csm complex has been described previously (15,30). The cyclase variant is unable to synthesise cOA signalling molecules due to mutation of the cyclase domain (15,30). Plasmid pCRISPR_TetR contains four identical spacers targeting the tetracycline-resistance gene and five repeats from M. tuberculosis (15,30). Plasmid pCRISPR which consists of two identical spacers targeting the pUC19 multiple cloning site (MCS) and three repeat sequences from M. tuberculosis was used for the ‘Not targeting crRNA’ control (15,30). Both plasmid pCRISPR_TetR and pCRISPR contain M. tuberculosis cas6. Plasmid pRAT containing the tetracycline-resistance gene without insert (i.e. without effector gene) was used as the ‘No Can2’ control (15,30). Plasmid pRAT-Duet containing the tetracycline-resistance gene has been described previously (30). Plasmid pRAT_TsuCan2 was constructed by cloning can2 from T. sulfidiphilus into the 5′-NcoI and 3′-SalI sites of the pRAT-Duet MCS-1 vector by restriction digest. The plasmid transformation assay was carried out essentially as described previously (15,30). E. coli C43 (DE3) cells containing pCsm1-5 ΔCsm6 and pCRISPRTetR were transformed by heat shock with 50 ng pRAT_TsuCan2 or pRAT plasmid, indicated as ‘Wild-type’ or ‘No Can2’ in Figure ​Figure3A,3A, respectively. E. coli cells containing pCsm1-5_Cy and pCRISPR_TetR were transformed with 50 ng of pRAT_TsuCan2 plasmid indicated as ‘Cyclase variant’ in Figure ​Figure3A.3A. E. coli C43 cells containing pCsm1-5 ΔCsm6 and pCRISPR were transformed with 50 ng of pRATTsuCan2 indicated as ‘No targeting crRNA’ in Figure ​Figure3A.3A. After outgrowth at 37°C for 2.5 h, 5 μl of a 10-fold dilution series was applied onto LB agar containing 100 μg ml−1 ampicillin and 50 μg ml−1 spectinomycin to determine the cell density of the recipient cells and onto LB agar additionally containing 25 μg ml−1 tetracycline, 0.2% (w/v) d-lactose and 0.2% (w/v) l-arabinose to determine the number of viable transformants. Plates were incubated at 37°C for 40 h. The experiment was carried out with two biological replicates and four experimental replicates each.Open in a separate windowFigure 3.Can2 protects against MGE in vivo. (A) Plasmid challenge assay of mycobacterial type III-A system in E. coli host. E. coli cells harbouring the mycobacterial type III-A interference complex Csm1-5 and TetR targeting spacer were transformed with plasmid containing TsuCan2 effector and tetracycline-resistance gene (Wild-type). Other strains are indicated as ‘No Can2’ where the TsuCan2 is absent, ‘Cyclase variant’ where the interference complex is unable to produce cOA and ‘No targeting crRNA’ where the TetR targeting spacer is replaced with a spacer targeting pUC19 MCS. A 10-fold dilution series of the transformation mixture was applied onto tetracycline selective plates to determine the number of viable transformants. (B) Growth curves of E. coli cells harbouring the interference complex and phage P1 lpa gene-targeting spacer supplemented with TsuCan2 effector (indicated as Wild-type). Cells were grown in LB broth in a 96-well plate with shaking at 37°C and infected with phage P1 at a MOI of ∼1. OD595 of the culture was measured every 15 min to plot against time over the 16 h incubation. Other strains are indicated as ‘Cyclase variant’ (green) where the interference complex is unable to produce cOA molecules; ‘No Lpa target’ (black) where lpa gene-targeting spacer is replaced with a spacer targeting pUC19 (MCS); ‘Can2 E302A/K304A’ (grey) which is a TsuCan2 nuclease variant. ‘Control’ (purple) represents wild-type cells (orange) incubated without phage infection under the same conditions. Data points represent the mean of eight experimental replicates (four biological replicates with two technical replicates each) with the standard deviation shown. (C) The OD595 values of all strains after 4 and 10 h growth are shown, coloured as in panel B. Statistical analysis was carried out with RStudio using the unpaired Welch two sample test to calculate P-values. NS (not significant) indicates P-values > 0.05 and *** indicates P-values < 1E–05.Bacteriophage infection assayA CRISPR array consisting of three identical spacers (Supplementary Table S2) targeting the lpa gene of bacteriophage P1 and four Mtb CRISPR repeats was ligated into the pCDFDuet™-1 vector (Novagen, Merck Millipore) to give pCRISPR_Lpa using the method described previously (15,30). T. sulfidiphilus (Tsu) can2 was cloned into the pEV5HisTEV vector between the NcoI and BamHI sites, and the nuclease domain variant E302A/K304A was generated using the QuikChange Site-Directed Mutagenesis kit as per manufacturer's instructions (Agilent Technologies). Plasmid pRAT_TsuCan2_E302A/K304A was constructed by cloning the variant gene from this pEV5HisTEV construct into the 5′-NcoI, 3′-SalI sites of pRAT-Duet MCS-1.Plasmids pCsm1-5 ΔCsm6, pCRISPRLpa and pRAT_TsuCan2 were co-transformed into E. coli C43 (DE3) cells indicated as ‘Wild-type’ in Figure ​Figure3B.3B. Plasmids pCsm1-5_Cy, pCRISPR_Lpa and pRAT_TsuCan2 were co-transformed into E. coli cells and are indicated as ‘Cyclase variant’ in Figure ​Figure3B.3B. Plasmids pCsm1-5ΔCsm6, pCRISPR and pRATTsuCan2 were co-transformed into E. coli cells and are indicated as ‘No Lpa target’ in Figure ​Figure3B.3B. Plasmids pCsm1-5ΔCsm6, pCRISPR_Lpa and pRAT_TsuCan2_E302A/K304A were co-transformed into E. coli cells and are indicated as ‘Can2 E302A/K304A’ in Figure ​Figure3B.3B. The cells were grown overnight at 37°C in LB broth containing 50 μg ml−1 ampicillin, 25 μg ml−1 spectinomycin and 12.5 μg ml−1 tetracycline. The overnight culture was diluted to OD600 of ∼0.1 (light path length: 10 mm) by LB broth supplemented with the antibiotics, 10 mM MgSO4, 0.2% (w/v) d-lactose and 0.2% (w/v) l-arabinose. 160 μl of diluted culture was infected with 40 μl bacteriophage P1 to give a MOI around 1 and was grown in a 96-well plate. The OD595 of the culture in the plate (light path length: ∼6.2 mm) was measured by a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices) every 15 min over 16 h incubation time. The experiment was carried out with four biological replicates and two technical replicates. The OD595 was plotted against time over the 16 h incubation.Co-crystallisation of Can2 in complex with cA4SthCan2 labelled with selenomethionine at 8 mg/ml was mixed with cA4 such that the molar ratio of protein:cA4 was 1:1. Crystallisation conditions were identified from the commercial screens JCSG and PACT 96 (Jena Biosciences). 75 μl of the mother liquor was added to the reservoir in a 96-well sitting drop plate, and the SthCan2 + cA4 solution was mixed with mother liquor in a 0.45 μl drop with a 2:1 or 1:1 protein:mother liquor ratio. Plates were incubated at room temperature. Initial screens yielded optimal crystals in 20% (w/v) PEG 3350 and 0.2 M ammonium nitrate pH 6.3, which required no further optimisation prior to data collection. Crystals were harvested into a fresh 1 μl drop of mother liquor and 1 μl glycerol was added to the drop for cryo-protection. Crystals were mounted on loops and vitrified in liquid nitrogen.X-ray data processing, structure solution, and refinementData were collected at Diamond Light Source (DLS) on beamline I03 at a wavelength of 0.9790–2.02 Å resolution (data processing and refinement statistics are shown in Supplementary Table S3). Diffraction images were automatically processed through the Xia2 pipeline (31) using XDS (32) and AIMLESS (33). Phasing information and structure solution was done using the automated experimental phasing pipeline BigEP (34) at DLS, which used SHELX (35) to assess the quality of the anomalous signal and CRANK (36) for phasing and to build the initial model. REFMAC5 (37) and COOT (38) were used for refinement of the model, and addition of ligands and water molecules. The cA4 ligand was drawn using Chemdraw (Perkin Elmer) and restraints generated in JLigand (39). Figures of the structures were created by CCP4mg (40) and PyMol (Schrödinger, LLC). The model was validated using tools in PDB-REDO (41) and Molprobity (42). The final Molprobity score is 1.2, centile 100, and Ramachandran statistics are 98.96% allowed, 0% disallowed. All structural alignment calculations were done using DALI (43). The coordinates and structure factors have been deposited in the Protein DataBank with accession code 7BDV.StatisticsStatistical analyses were performed with GraphPad Prism. Statistical significance was assessed as described for each experiment.

Article TitleThe CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence

Abstract

The molecular coordinates for the Can2 protein structure are available in the Protein Databank under accession code 7BDV.


Login or Signup to leave a comment
Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.