Materials and Methods

Interference activity of a minimal Type I CRISPR–Cas system fromShewanella putrefaciens

MATERIALS AND METHODSBacterial strains and growth conditionsBacterial strains and plasmids used in this study are listed in Supplementary Table S1. E. coli strains DH5α, DH5α λpir and WM3064 were grown in LB medium at 37°C. Media for the growth of the 2,6-diamino-pimelic acid (DAP)-auxotroph E. coli WM3064 strain were supplemented with 300 μM of DAP. E. coli strain BL21(DE3) pLysS was grown either in LB or in NZ-amine (1% NZ-amine, 0.5% yeast extract and 1% NaCl) media at 37°C until an OD600 of ∼0.6 was reached. Protein expression was induced by addition of 1 mM Isopropyl β-d-thiogalactopyranoside (IPTG) and continuous growth at 18°C. S. putrefaciens CN-32 was cultured in LB medium at 30°C. Media were supplemented with 50 μg/ml spectinomycin, 50 μg/ml kanamycin, 50 μg/ml ampicillin or 10% (w/v) sucrose.Construction of cas gene deletion strainsS. putrefaciens CN-32 mutants with markerless in-frame deletions or integrations were constructed using the primers listed in Supplementary Table S2 and the suicide vector pNPTS138-R6KT (KanR) following the procedure reported in (45). Briefly, appropriate ∼500 bp homologous fragments of the up- and downstream regions of the target gene were amplified. For deletions, the codons encoding the first and last four amino acids were retained. The resulting PCR-derived DNA fragments were joined via overlap PCR, treated with appropriate restriction enzymes and ligated with the equally digested pNPTS138-R6KT vector. The resulting plasmids were sequenced and transferred to S. putrefaciens CN-32 by conjugation from E. coli WM3064. Target gene regions were replaced by the corresponding modified version via sequential homologous crossover and correct deletions were identified via colony PCR using appropriate primers.Point mutations were introduced into the S. putrefaciens CN-32 gene Sputcn32_1822. To this end, the wild-type gene sequence, along with 500 bp of upstream and downstream sequences was cloned into pNPTS138-R6KT. Point mutations were introduced into this plasmid by QuickChange site-directed mutagenesis (Stratagene) using primers that were designed using the Agilent QuickChange Primer Design Tool. The plasmids carrying the point mutations were applied for in-frame insertions in S. putrefaciens CN-32 as described above.RNA isolation, sequencing and Northern blot analysisNucleic acids were extracted from an overnight culture of S. putrefaciens CN-32 using phenol/chloroform (1:1 phenol pH 5 for RNA and pH 8 for DNA). The mirVana RNA extraction kit (Ambion) was used to isolate small RNAs (<200 nt) from total RNA. To ensure proper termini for adapter ligation, 1 μg of the small RNA preparation was incubated with 10 U of T4 polynucleotide kinase (T4 PNK, Ambion) at 37°C overnight, followed by 1 h incubation at 37°C in presence of 1 mM ATP. This treatment ensures 5′-monophosphate termini suitable for RNA-Seq library preparations. RNA libraries were sequenced by Illumina HiSeq2000 sequencing at the Max-Planck Genome Centre, Cologne.Northern blot analyses required the extraction of 10 μg of total RNA from wild-type and cas gene-knockout strains of S. putrefaciens CN-32. The RNA preparations were denatured (95°C for 5 min) in formamide loading buffer and separated via electrophoresis on a 10% TBE–8 M urea polyacrylamide gels at 200 V for 1 h. The separated bands were transferred to a nylon membrane (Roth) and subjected to UV-crosslinking at 25 V for 2 h. DNA probes were radiolabeled with 5′-γ32P-ATP using T4 PNK and incubated with the membrane, which was pre-incubated at 42°C for 30 min with ULTRAhyb-Oligo buffer (Ambion). After overnight hybridization at 42°C, the membranes were washed two times for 15 min with 2× SSC buffer and 0.1% SDS and with 0.1× SSC and 0.1% SDS. Radioactive signals were detected by phosphorimaging.Conjugation-assays for DNA interference analysisTarget plasmids for conjugation assays were designed with the primers listed in Supplementary Table S2. Individual primer pairs were hybridized generating the sticky ends of EcoRI and BamHI restriction sites that flanked the protospacer sequences. The primer pairs were phosphorylated at the 5′-termini by T4 PNK, mixed and hybridized (95°C for 5 min, followed by slow cooling to room temperature). The obtained DNA fragments were ligated into a linearized pBBR1MCS2 (KanR) vector. Insertion of the fragment was confirmed via sequencing.The obtained plasmids were transformed into the donor strain E. coli WM3064. Equal amounts of E. coli and S. putrefaciens CN-32 strains overnight cultures were harvested. The strains were suspended in 100 μl of DAP-supplemented LB medium and mixed before spotting a single drop on nonselective LB agar supplemented with DAP. After overnight incubation at 30°C, the cells were washed from the plate using 2 ml of nonselective LB. The mating mixture was then washed three times with 1 ml of nonselective LB and screened for conjugated S. putrefaciens CN-32 by plating 100 μl of the mating mixture on LB plates supplemented with Kan but lacking DAP. This procedure was performed in triplicate and in parallel for the control plasmid without protospacer sequences. The number of obtained colonies was counted for conjugation of the plasmid with protospacer (pT) and the control plasmid (pNT). Relative conjugation efficiency was calculated as pT/pNT ratio and the transconjugant counts are detailed in Supplementary Table S3.Production and purification of recombinant proteinsThe cas genes of S. putrefaciens CN-32 were amplified from genomic DNA and cloned into gene expression vectors. The cas6f gene was cloned into pET20b (AmpR) to generate Cas6f with a C-terminal His-tag. The gene cassette cas1821, cas1822, cas6f was cloned into pRSFDuet; this construct allows the simultaneous production of all three proteins with only Cas1821 having an N-terminal His-tag fusion. The individual cas1821 gene was cloned to produce a His-Sumo-tag fusion that was processed as previously described for Thermoproteus tenax Cas proteins (22). Cas6f production was induced by addition of 1 mM IPTG followed by overnight growth at 18°C of the E. coli BL21(DE3)pLysS host strain. Cell lysates were pepared by sonication in a buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM dithiothreitol (DTT), 0.01% Triton X100 and 20 mM imidazole. Proteins were purified on a HiTrap Ni-NTA column (GE Healthcare) in a linear imidazole gradient ranging from 100 to 500 mM imidazole. The Cas1821 protein was subjected to Ni-NTA chromatography in a buffer containing 100 mM potassium phosphate pH 7.5, 500 mM NaCl, 10% glycerol. A high-salt washing step with a buffer including 1 M NaCl was added before protein elution to remove nucleic acid contaminants. The SUMO tag was cleaved using SUMO protease during overnight dialysis at 4°C in a buffer containing 50 mM Tris–HCl pH 7.0, 100 mM NaCl and 10% glycerol. The protein was further purified over a HiTrap Heparin Sepharose HP column and size exclusion chromatography on a Superdex 200 column.The production of recombinant Cascade required the presence of the pRSFDuet vector containing the genes cas1821, cas1822, cas6f and a pUC19 vector containing the repeat-spacer4-repeat sequence from the CRISPR array cloned downstream of a T7 RNA polymerase promoter. The 100 nt transcript is processed by Cas6f into a 60 nt crRNA. BL21(DE3)pLysS cells with both plasmids were grown in the presence of ampicillin, kanamycin and spectinomycin. Protein and pre-crRNA production was induced at an OD600 of 0.6 by addition of 1 mM IPTG, followed by overnight growth at 18°C. Cascade crRNP complexes were purified via Ni-NTA chromatography in a buffer containing 50 mM Tris-HCl pH 7.0, 500 mM NaCl, 10% glycerol, 1 mM DTT and 20 to 500 mM imidazole. Size exclusion chromatography was performed on a Superdex 200 column (calibrated with a molecular weight standard kit (12 000–200 000 Da, Sigma-Aldrich)) in a buffer containing 50 mM HEPES-NaOH pH 7.0, 150 mM NaCl and 1 mM DTT.Transmission electron microscopy of Cas1821Purified protein samples were negatively stained with 2% (w/v) uranyl acetate as described previously (22,46,47). Subsequent electron microscopy was carried out with a JEOL JEM-2100 transmission electron microscope equipped with a LaB6 cathode at 120 kV (JEOL, Tokyo, Japan). Electron micrographs were taken with a 2k × 2k fast-scan CCD camera F214 combined with EM-Menu 4 (TVIPS, Gauting, Germany).

Article TitleInterference activity of a minimal Type I CRISPR–Cas system fromShewanella putrefaciens

Abstract

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species includingShewanella putrefaciensCN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassifiedcasgenes. The genome ofS. putrefaciensCN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is activein vivoand that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion ofcas1821andcas1822reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.


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.