MATERIALS AND METHODSStrains and growth conditionsThe strains and plasmids used in this study are listed in Supplemental Table S1. Pfu strains were grown under strict anaerobic conditions at 90°C in defined medium. The medium was composed of 1× base salts, 1× trace minerals, 10× vitamin solution, 2× 19-amino-acid solution, 10 μM sodium tungstate, 0.35% cellobiose, and 1 mg/l resazurin, with added cysteine at 1 g/l, sodium sulfide at 0.5 g/l, sodium bicarbonate at 1 g/l and 1 mM sodium phosphate buffer (pH 6.5). Stock solutions were prepared as described previously (52) with the exception that vitamin solution was prepared as a 4000× solution rather than 200× and cysteine was used instead of cysteine–HCl. Medium pH was adjusted before the addition of phosphate buffer to approximately pH 6.5. Liquid cultures were inoculated with 1–2% inoculum or with a single colony and grown in anaerobic culture bottles. Anaerobic culture bottle headspace was exchanged with four cycles of vacuum and argon. Solid media preparation and culture growth was as described previously (52). Medium was supplemented with 20 μM uracil and/or 2.75 mM 5-FOA as needed for selection. E. coli strain TOP10 was used for general plasmid DNA manipulation. Cultures were grown at 37°C in Luria Broth (Millers) supplemented with apramycin sulfate (50 μg/ml).Northern analysisTotal RNA samples were isolated from ∼50 mg of Pfu cells (JFW02, TPF07, TPF15, TPF17 or TPF20) using Trizol LS (Invitrogen). Ten microgram of total RNA samples were separated on 15% TBE–urea polyacrylamide gels beside a 32P 5′ end-labeled Decade Marker RNA (Life Technologies). RNAs were transferred to nylon Zeta-Probe membranes (Bio-Rad) using a Trans-Blot SD Semi-Dry Cell (Bio-Rad). Membranes were baked at 80°C for an hour before prehybridization in a ProBlot hydridization oven (LabNet) for an hour at 42°C. Prehybridization and hybridization were performed in hybridization buffer containing 5× SSC, 7% SDS, 20 mM NaPO4 (pH 7.0) and 1× Denhardt's solution. Deoxyribonucleotide probes (Operon) (10 pmol) were 5′ end-labeled with T4 Polynucleotide Kinase (NEB) and γ-32P-ATP (specific activity > 6000 Ci/mmol, Perkin Elmer) using standard protocols. Labeled probes (1 million cpm/ml) were added to prehybridization buffer, followed by hybridization at 42°C overnight. Probed membranes were washed twice in 2× SCC, 0.5% SDS for 30 min at 42°C. Membranes were reprobed for the 5S rRNA loading control following initial probing. Radioactive signals were detected by phoshorimaging (Storm 840 Scanner GE Healthcare). Probe sequences used are listed in Supplemental Table S2.General DNA manipulation and plasmid constructionPlasmid DNAs for sequencing and routine analysis were isolated from E. coli strain Top10 using the QIAprep Spin Miniprep Kit (Qiagen). Large-scale plasmid DNA isolation from the Top10 strain was carried out for the construction of plasmids or for use in Pfu plasmid transformation assays, using the Zyppy Plasmid Maxiprep Kit (Zymo Research). Following isolation with the Maxiprep kit, plasmids were isopropanol-precipitated and resuspended in 1 mM Tris–Cl, pH 8.5. Routine PCR screening was carried out with Crimson Taq (NEB), and Splicing by Overlap Extension PCR (SOE-PCR) was carried out with Phusion polymerase (NEB). Pfu genomic DNA was isolated from 1 ml of overnight liquid cultures with the Zymo Quick gDNA Miniprep kit (Zymo Research) for routine PCR genotyping.The expression plasmid pJE47 was constructed using the T. kodakarensis csg promoter and chiA terminator sequences found on the plasmid pLC64-ChiA (6). Primers (sequences given in Supplemental Table S2) Pcsg_F and Pcsg_NdeI_Bam_R were used to amplify the promoter region, and primers Term_NdeI_Bam_F and ChiA_Term_R were used to amplify the terminator region. The PCR products were spliced by SOE-PCR, in the process adding NdeI and BamHI restriction sites between the promoter and terminator, and cloned into the NotI/EcoRV sites of the Pfu shuttle vector plasmid pJFW18 (53).Plasmids containing CRISPR target inserts (Supplemental Table S1) were constructed by one of three methods. Plasmids pJE18–33 were constructed by ligating annealed 5′-phosphorylated oligos (Supplemental Table S2) into the NotI site of pJFW18. Plasmid was linearized with NotI-HF (NEB) and dephosphorylated with thermosensitive alkaline phosphatase (Promega). 5′-phosphorylated CRISPR target oligo pairs were annealed in 10 mM Tris–Cl (pH 8), 1 mM EDTA, 500 mM NaCl. Annealed oligo pairs were ligated with NotI-linearized pJFW18 using T4 DNA Ligase (NEB).Plasmids pJE186-249 were constructed by a combination of two methods. The inserts for the majority of the plasmids were constructed by primer extension of oligo 7.01_TIM_Sat_Mut_F, which contains degenerate PAM nucleotides, with primer 7.01_TIM_Sat_Mut_R. The primer extended oligo pairs were digested with NdeI (NEB) and BamHI-HF (NEB) and ligated with NdeI/BamHI-linearized pJE47. After analysis of ∼200 clones, all remaining tri-nucleotide combinations were cloned into pJE47 as described for plasmids pJE18–33, with the exception that the host vector was NdeI/BamHI-linearized pJE47. Additional 6.01 target plasmids, pJE275 & pJE299–301, were generated by ligation of annealed 5′-phosphorylated oligos with NdeI/BamHI-linearized pJE47.Plasmids pJE252 (Cas3), 253 (Cas3”), and 269 (Cas3′+3”) were constructed using standard cloning techniques. Inserts for these plasmids were generated by PCR amplification of the genes using JFW02 genomic DNA. Each insert was generated using the following primer sets: Cas3 with PF1120_pJE47_F/R, Cas3” with PF0639_pJE47_F/R, and Cas3′+3” with PF0640_pJE47_F/PF0639_pJE47_R. Inserts were cloned into NdeI/BamHI linearized pJE47 with the GENEART Seamless Cloning and Assembly kit (Life Technologies). To generate plasmids pJE257, 258 and 270, these plasmids were linearized and dephosphorylated with NotI-HF and thermosensitive alkaline phosphatase prior to ligation with annealed PF_7.01_GGG+/- oligos.All E. coli transformants were analyzed by PCR for inserts and sequenced to confirm insert sequence and orientation. Primer sequences used for PCR amplification of inserts and oligo pairs used for the construction of target-bearing plasmids are indicated in Supplemental Table S2, with the ‘+’ oligo being annealed with the cognate ‘-’ oligo to generate a double-stranded target for ligation.Pyrococcus furiosus strain constructionPfu strains were constructed to characterize individual effector complexes in vivo, using a variant of the previously described pop-in/pop-out marker replacement technique (Supplemental Figure S4) (52,54). Linear PCR products containing a counter-selectable pyrF wild type allele were transformed into Pfu host strains containing a deletion of the pyrF gene to select uracil prototrophy. Regions of homology flanking the pyrF gene were used to guide homologous recombination at desired genomic regions. Following marker replacement of the region of interest, 5-FOA, a toxic PyrF substrate, was used to select for cells that had removed the pyrF wild type allele by homologous recombination between short regions of homology, internal to the PCR fragment, flanking the pyrF gene resulting in a markerless deletion of the gene of interest. The transformed PCR products were generated by splicing four PCR products together with SOE-PCR (Supplemental Figure S4 and Supplemental Table S2). Strain TPF07 (Csa) (Supplemental Table S1) was constructed by a single-step deletion of genes PF1130 through PF1121 (Δcmr+cst). TPF15 (Cmr), TPF17 (Cst) and TPF20 (null) strains were each constructed with two consecutive deletion steps. TPF15 (Cmr) was constructed by stepwise deletions with the Δcsa (deletion of PF0637 through PF0644) and Δcst (deletion of PF1123 through PF1121) PCR constructs. TPF17 (Cst) was constructed with the Δcmr (deletion of PF1130 through PF1124) and Δcsa constructs. TPF20 (null) was constructed with the Δcmr+cst and Δcsa PCR constructs. TPF01 (ΔCas3”) and TPF02 (ΔCas3) strains were constructed by deletion of PF0639 and PF1120, respectively, from the parental strain JFW02. TPF10 (ΔCas3”, ΔCas3) was generated by deletion of PF1120 from TPF01. TPF29 (Cst ΔCas3) was generated by deletion of PF1120 from TPF17, and TPF30 (Csa ΔCas3”) was generated by deletion of PF0639 from TPF07.Plasmid transformation assay in Pyrococcus furiosusPlasmid transformations were accomplished by culturing Pfu strains anaerobically at 90°C to mid-to-late log phase in defined media supplemented with 20 μM uracil. 33.3 ul of the culture was mixed with plasmid DNA to a final concentration of 2.0 ng/μl (35 μl total reaction volume) and briefly incubated at room temperature for typically 5–60 min before plating on solid defined media lacking uracil. The mixture was spread on solid media using Coliroller glass beads (EMD Millipore) and incubated for ∼64 h at 90°C under anaerobic conditions. Following incubation, colonies per plate were enumerated. All transformation assays were carried out with a minimum of three technical replicates.
CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids.Pyrococcus furiosus(Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. ThePfuCmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system inPfustrains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci inPfuare processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.