Materials and Methods

Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system

MATERIALS AND METHODSCloning, gene expression and protein purificationCas6-1 (Sso1437) was expressed and purified as described previously (14). The gene for Cas6-3 (sso1422) was amplified by PCR and cloned using NcoI and EcoRI sites into a derivative of the vector pEHisTEV (22), allowing expression with an N-terminal polyhistidine-tagged maltose binding protein (MBP) fusion. The K47A variant was constructed by site directed mutagenesis using the Quikchange protocol (Stratagene). The sequences of the PCR and mutagenesis oligonucleotides are available from the corresponding author on request. MBP-Cas6-3 protein was expressed in Escherichia coli C43 cells by induction at an O.D.600 of 0.8 with 0.4 mM isopropyl β-D-1 thiogalactopyranoside (IPTG) and overnight incubation at 25°C with shaking. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 30 mM imidazole, 10% glycerol), disrupted by sonication, centrifuged at 30 000 × g for 20 min and the supernatant filtered through a 0.2 μM filter. The cleared lysate was subjected to immobilized metal affinity chromatography on a 5 ml HisTrap FF column equilibrated with buffer A and eluted with a gradient of 30–500 mM imidazole in buffer A. Fractions containing MBP-Cas6-3 were pooled and applied to a Superdex 200 10/300 column equilibrated with buffer B (20 mM Tris–HCl pH 7.5, 300 mM NaCl, 10% glycerol) and eluted by isocratic flow in the same buffer. The final protein eluted in roughly equimolar quantities with the E. coli chaperone GroEL, suggesting problems with correct protein folding. The solubility of the protein was dependent on the presence of the MBP tag. These limitations meant that the MBP-Cas6-3 protein was only suitable for simple qualitative activity assays.Sequence and preparation of RNA substratesRNA was purchased from IDT. The CRISPR repeats corresponding to S. solfataricus P2 CRISPR repeats of the C and D (CD) and A and B (AB) loci were synthesized in both fluorescently labelled (5′-fluorescein (FAM)) and unlabelled formats. An extra uracil nucleotide was added to the 5′ end of the repeat oligonucleotides to prevent fluorescence quenching by the guanine nucleotide that is the first bona fide nucleotide of both the AB and CD-repeat families. As a mimic of the Cas6 cleavage product, a fluorescently labelled CD sequence lacking the final eight 3′ nucleotides (CDproduct) was synthesized with a 3′-phosphate. The predicted Cas6 substrate leading to the generation of ncRNA60 was synthesized in an unlabelled format. The non-fluorescent oligonucleotides were end-labelled with γ-32P-ATP and polynucleotide kinase and purified as described previously (23)AB: 5′-GAUUAAUCCCAAAAGGAAUUGAAAGCD: 5′-GAUAAUCUCUUAUAGAAUUGAAAGncRNA60: 5′-UAAUGUGCCCCAAAAUGAAUUGAUAUAB: 5′-FAM-UGAUUAAUCCCAAAAGGAAUUGAAAGCD: 5′-FAM-UGAUAAUCUCUUAUAGAAUUGAAAGCDproduct: 5’-FAM-UGAUAAUCUCUUAUAGA-PSingle-turnover assaysTwo micromolars purified recombinant Cas6 was incubated with 1–5 nM γ-32P ATP-labelled RNA (CD, AB or ncRNA60) at 60°C in nuclease reaction (NR) buffer (20 mM NaH2PO4/Na2HPO4­ pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5 mM dithiothreitol). This buffer supported a higher Cas6 activity than that reported previously (14). To stop the reaction, aliquots of 10 μl were quenched by addition to 30 μl acid phenol/chloroform (Ambion), vortexed briefly and centrifuged at 15 000 × g for 1 min. Five microlitres of the upper aqueous phase was removed and mixed 1:1 with formamide. Samples were heated at 95°C for 2 min and loaded onto a pre-run 20% denaturing polyacrylamide gel (20% acrylamide, 8 M urea, 1× Tris-borate-EDTA (TBE)) then electrophoresed at 80 W for 90 min in 1× TBE running buffer. Following electrophoresis, gels were scanned by phosphorimaging and analysed using Fuji Imagegauge software as described previously (24).Multiple-turnover assaysCas6-1 (4–80 nM) was incubated with a large excess (4 μM) CD RNA in NR-buffer additionally supplemented with 2 μM bovine serum albumin (BSA). All other details were as described above.Electrophoretic mobility shift assayA dilution series of Cas6-1 was prepared from 5 μM to 0.5 nM (monomer concentration) in NR buffer supplemented with 2 μM BSA, to which 0.5 nM γ-32P ATP-labelled RNA was introduced. After 15 min incubation at 20°C, reactions were gently mixed 1:1 with ficoll and 10 μl loaded under voltage (30 V) onto a non-denaturing­ 6% polyacrylamide, 1× TBE gel. Electrophoresis was completed at 120 V for 120 min in 1× TBE running buffer. Gels were phosphorimaged, quantified to determine the fraction of RNA bound and plotted using Kaleidagraph as detailed previously (23).Fluorescence quenchingFluorescence quenching experiments utilized a Varian Cary Eclipse fluorimeter (λexcitation = 480 nm) under temperature control at 25°C. In a 150 μl starting volume, changes to fluorescence intensity were recorded on addition of aliquots of Cas6-1 to 1 nM CD or CDproduct oligonucleotide with a 5′ fluorescein label. The emission data collected in the wavelength range 500–560 nm (1 nm data intervals) were summed, adjusted for dilution factor and normalized for baseline fluorescence. Assays were conducted in NR buffer supplemented with 2 μM BSA.Determination of Cas6 activity in size-fractionated lysates of S. solfataricusFive grams S. solfataricus was grown, pelleted and lysed as described previously (19), resuspended in GF buffer (20 mM NaH2PO4/Na2HPO4­ pH 7.5, 250 mM NaCl, 5 mM ethylenediaminetetracetic acid (EDTA), 0.5 mM dithiothreitol (DTT), filtered through a 0.2 μm filter and applied to a Superdex 200 gel filtration column (GE Healthcare) equilibrated in GF buffer. Protein was eluted isocratically and 2 ml fractions were collected. Cas6 activity was determined as described above for single turnover conditions using a single time point of 60 min at 60°C. Aliquots were also analysed by western blotting using a polyclonal sheep antibodies raised against the Cas5–Cas7 (Sso1441–1442) complex of S. solfataricus Cascade, the Cmr7 (Sso1986) subunit of CMR and the Cas6-1 protein (SNBTS, Penicuik, Midlothian), using standard methods. In a separate experiment, recombinant Cas6-1 was passed through the same column under the same conditions in GF buffer.

Article TitleCas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system

Abstract

CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. Highly studied CRISPR-Cas systems such as those inEscherichia coliandPseudomonas aeruginosahave a single Cas6 enzyme that is an integral subunit of the surveillance complex. By contrast,Sulfolobus solfataricushas a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues fromS. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes reveals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling between Cas6 paralogues and their downstream effector complexes.


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