MATERIALS AND METHODSStrains and plasmidsStrains and plasmids used in this work are listed in Supplementary Tables S1 and S2, respectively. The strains producing crRNA with altered spacer length are derivatives of KD263 (20) and were constructed using a technique based on the previously published Red recombinase protocol (21). Synthetic dsDNA fragments (gBlocks from IDT Inc.) containing CRISPR arrays with appropriate spacer lengths were used for recombination.The target plasmids pG8 and pG8mut are pT7blue-based plasmids containing, respectively, a wild-type protospacer (5′-CTGTCTTTCGCTGCTGAGGGTGACGATCCCGC) and preceding ATG PAM and a mutant variant carrying the C1T substitution at the first position of the protospacer (22). The protospacer is followed by a 5′-ATGTAT-3′ sequence matching the extension in the +6 spacer.For in vitro studies of Cascade complexes assembled on crRNAs with altered-length spacer, the pCDF-based plasmids co-expressing cse2, cas7, cas5 and cas6e genes and appropriate CRISPR arrays were generated. DNA fragments containing CRISPR arrays with altered spacer length were PCR amplified using genomic DNA isolated form appropriate strains and the following primers: XhoI-forward (5′-ACCCCTCGAGATTTGGATGGTTTAAGGTTGGTG-3′) and AvrII-reverse (5′-AGGCCTAGGCGAAGGCGTCTTGATGGG-3′) and cloned into XhoI and AvrII sites of the pCDF-casBCDE plasmid (23).RNA extraction and Northern blottingThe procedure was performed as described previously (23). 32P-end labeled probe (5′-GCAGCGAAAGACAGCGGTTTAT-3′) complementary to the 5′-part of all crRNA spacer-length variants was used for hybridization.CRISPR interference and adaptation assaysE. coli strain KD263 (20) carrying a genomic CRISPR array with 32-nucleotide (wt) spacer, strain KD390 (23) harboring a single repeat in CRISPR array or derivative strains (KD692, KD696, KD698, KD700, KD702) harboring CRISPR arrays containing altered-length spacers were used to determine cell sensitivity to M13 phage infection by a spot test method as described (24). Efficiency of plaquing was calculated as a ratio of the number of plaques formed on a lawn of tested cells to the number of plaques on sensitive (non-targeting, KD390) cell lawn. For each strain, plaquing efficiency was determined in at least three independent experiments.For plasmid transformation efficiency assay, competent pre-induced cells were prepared and electroporated with 10 ng of pG8, pG8mut or control pT7blue plasmids. Transformation efficiency was determined as ampicillin-resistant colony numbers per microgram DNA. Mean values and standard deviations were obtained from three independent experiments.To analyze plasmid loss, individual colonies of uninduced cells transformed with pG8 or pG8mut plasmids were inoculated in 3 ml LB broth supplemented with 100 μg/ml ampicillin for overnight growth at 37°C. Aliquots of overnight cultures were diluted 100-times into fresh LB without antibiotic and allowed to grow until the culture OD600 reached 0.5. At this point half of the culture was induced with 1 mM arabinose and 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Another half of the culture remained uninduced. Plasmid DNA was purified from 1.5-ml aliquots withdrawn at various times post-induction using Thermo Scientific GeneJET Plasmid Miniprep Kit. Purified plasmids were analyzed on 0.9% agarose gels.To monitor CRISPR adaptation, KD263 or strains producing crRNA with altered spacer length transformed with the pG8, pG8mut or pT7blue plasmids were grown overnight at 37°C in LB broth supplemented with 100 μg/ml ampicillin. Aliquots of cultures were diluted 100-fold with LB broth without ampicillin, allowed to grow until OD600 1.0 and then supplemented with 1 mM arabinose and 1 mM IPTG and grown overnight. To monitor spacer acquisition 1 μl of culture was added to 20 μl polymerase chain reaction (PCR) with primers Ec_LDR-F (5′- AAGGTTGGTGGGTTGTTTTTATGG-3′) and Ec_minR (5′- CGAAGGCGTCTTGATGGGTTTG-3′). PCR products corresponding to expanded CRISPR cassettes were gel purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced with MiSeq Illumina System at Moscow State University Genomics facility.High throughput sequence analysisRaw sequencing data were analyzed using ShortRead and BioStrings packages (25,26). Illumina-sequencing reads were filtered for quality scores of ≥20 and reads containing two repeats (with up to two mismatches) were selected. Intervening sequences were considered as spacers. Spacers were next mapped on the pG8 or pG8mut plasmids with no mismatches allowed. R scripts and their package ggplot2 (27) were used for spacers statistics. Circle histograms of spacer mapping were obtained with EasyVisio tool created by E. Rubtsova.Cascade expression and purificationCascade subcomplexes lacking Cse1 were prepared from E. coli KD418 cells (28) co-expressing cas genes and appropriate CRISPR cassette from pCDF-based plasmids. Cascade subcomplexes containing N-terminal Strep-Tag II fused to Cse2 subunit were affinity-purified on Strep-Tactin® column (IBA) from cells grown at 37°C until OD550 reached 0.5 followed by 4-h induction with 1 mM IPTG. Purification buffers contained 100 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM β-mercaptoethanol and 1 mM ethylenediaminetetraacetic acid (EDTA). Binding buffer additionally contained 0.1 mM phenylmethanesulfonyl fluoride and Elution buffer contained 2.5 mM desthiobiotin and 1 mM (tris(2-carboxyethyl)phosphine) TCEP. Complexes were further separated using a Superdex 200 HiLoad 16/60 column (Amersham Biosciences) equilibrated by 50 mM Tris-HCl, pH 8, containing 150 mM NaCl, 1 mM EDTA and 1 mM TCEP.N-terminally 6His-tagged Cse1 was purified by IMAC from E. coli KD418 strain transformed with an appropriate expression plasmid followed by gel-filtration on Superdex 200 HiLoad 16/60 column (Amersham Biosciences) equilibrated with 20 mM HEPES-K buffer (pH 7.5) containing 150 mM NaCl.crRNA analysisTo verify molecular size of crRNA 1 μl of Cascade samples was added to 10 μl reaction mixture containing 2.5 μCi γ-32P-ATP (6000 Ci/mmol), 5 units of T4 polynucleotide kinase (PNK, New England Biolabs) and 1x PNK reaction buffer (New England Biolabs). After 30 min incubation at 37°C, reactions were stopped with equal volume of formamide-containing loading buffer, boiled for 1 min and products resolved by denaturing 8 M urea, 20% polyacrylamide gel electrophoresis (PAGE) and revealed by autoradioagraphy.Native gel analysisCascade samples were loaded on a native precast 4–12% gradient polyacrylamide gel (NovexTM, Invitrogen) and run in Tris-Glycine buffer at 25 mA for 2 h. Protein bands were visualized by staining with SimplyBlueTM (Invitrogen) according to manufactorer's protocol. To identify protein bands carrying crRNA, Cascade samples were incubated with 32P-end labeled oligonucleotide probe complementary to the 5′-part of crRNA (the same probe was used for Northern blotting) in the binding buffer (40 mM Tris-HCl, pH 8.0, 50 NaCl, 5 mM MgCl2, 0.5 mM TCEP, 50 μg/ml bovine serum albumin (BSA)) at room temperature for 15 min prior to loading on native gel. Products were revealed by autoradiography.Protein and native mass spectrometryCascade complex samples were sprayed from in-house prepared gold-coated borosilicate glass capillaries and analyzed on a SYNAPT G2-Si instrument (Waters) as reported before (18). Briefly, purified Cascade complexes were buffer exchanged to 100 mM ammonium acetate, pH 7 (Sigma) using 3 kDa molecular weight cutoff spin filters (Pall Corp.) and infused to electrospray source at protein concentration of 2–3 μM and the rate around 90 nl/min. To assure best instrument performance in high mass-to-charge range critical parameters were adjusted as follow: source temperature 30°C, capillary voltage 1.7 kV, trap bias voltage 16 V and argon pressure in collision cell (trap) 7 ml/min. Transfer collision energy was kept at constant level of 10 V while trap energy varied between 10–200 V. To obtain accurate mass measurement for individual protein subunits, Cascade samples were dissociated and completely unfolded using a 1% of formic acid (Sigma) in acetonitrile solution mixed with complex in 1:1 ratio. Data analysis was performed in MassLynx software version 4.1 (Waters).Permanganate probingTarget dsDNA 209-bp fragment was PCR amplified from plasmid pG8 with g8-F 5′-CTTTAGTCCTCAAAGCCTCTG-3′ and g8-R 5′-GCTTGCTTTCGAGGTGAATTTC-3′ primers. The 5′ ends of the target DNA fragment were labeled with 32P using T4 PNK for 30 min at 37°C (5–10 pmoles of 5′ termini in 30 μl reaction mixture containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DTT, 10 pmoles γ-32P-ATP (6000 Ci/mmol) and 10 units of T4 PNK (New England Biolabs). 32P-labeled DNA fragments were purified on micro Bio-SpinTM chromatography columns packed with Bio-Gel P-6 (Bio-Rad). KMnO4 probing was performed with 5 nM labeled DNA fragment, 0.2–0.4 mM of Cascade complex, in 10 μl binding buffer (40 mM Tris-HCl, pH 8.0, 50 NaCl, 5 mM MgCl2, 0.5 mM TCEP, 50 μg/ml BSA). Before probing, the Cse2Cas7Cas5Cas6e-crRNA subassemblies were incubated with Cse1 (1:1.5 molar ratio) in binding buffer at 37°C for 15 min. After the addition of DNA, incubation at 37°C was continued for 30 min and probing reaction was initiated by adding KMnO4 to a final concentration of 2.5 mM. Reactions were incubated for 15 s at 37°C, quenched by the addition of 10 μl of 1% β-mercaptoethanol, followed by 5 μg of calf thymus DNA in 50 μl of 10 mM Tris-HCl (pH 8.5). Reactions were extracted with phenol–chloroform mixture, followed by ethanol precipitation. DNA pellets were dissolved in 100 μl of freshly prepared 1 M piperidine and heated in dry bath at 95°C for 20 min. Piperidine was removed by chloroform extraction and DNA was ethanol precipitated. Pellets were dissolved in 10–15 μl of formamide loading buffer and products were separated by denaturing 8% PAGE and revealed by autoradiography.Cas3 cleavageCas3 digestion of DNA in preformed complexes with target DNA was performed in 10 mM HEPES pH 7.4, 60 mM KCl, 10 mM MgCl2, 20 μM CoCl2, 2 mM adenosine triphosphate (ATP) by 800 nM Cas3 for 50 min at 37°C. The reactions were stopped with 2 volumes of formamide-loading buffer, heated at 100°C for 1 min and products resolved by denaturing 8 M urea, 10% PAGE.Fluorometric measurementsCascade beacons were formed by mixing oligonucleotide 1 labeled with fluorescein at 5′ end, unmodified oligonucleotide 2 and oligonucleotide 3 labeled with Iowa Black® FQ at 3′ end (final oligonucleotide concentrations were within low μM range) in a buffer containing 40 mM Tris, pH 7.9, 100 mM NaCl by heating for 1 min at 90°C and slow cooling to 20°C. Oligonucleotides 2 and 3 were taken in 30% excess to oligonucleotide 1 to avoid the presence of free oligonucleotide 1 in samples. Control experiments verified that such excess of the oligonucleotides 2 and 3 had no effect on beacon binding.Fluorescence measurements were performed using a QuantaMaster QM4 spectrofluorometer (PTI) in binding assay buffer (20 mM Tris HCl (pH 7.9), 50 mM NaCl, 5% glycerol, 0.1 mM DTT and 5 mM MgCl2) containing 0.02% Tween 20 at 25°C. Final assay mixtures (800 μl) contained 40 or 80 nM of a Cascade-crRNA complex and 2 nM Cascade beacon construct. The fluorescein fluorescence intensities were recorded with an excitation wavelength of 498 nm and an emission wavelength of 520 nm. Time-dependent fluorescence changes were monitored after addition of negligible volume of Cascade beacon to a cuvette followed by manual mixing; the mixing dead-time was 15 s.Analysis of Type I spacers from public databaseCRISPRFinder (17) with default parameters were used to fetch all CRISPR arrays from archaeal and bacterial sequences downloaded from NCBI FTP site (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/). A total of 488 437 spacers were found. CRISPR-Cas system types were annotated using procedures described in (1,29). Only one CRISPR array per CRISPR-Cas subtype per species Tax ID were taken resulting in a set of 3214 spacers for Type I CRISPR-Cas systems. R library ggplot2 (27) was used to generate histograms for spacer length distribution.
Article TitleAltered stoichiometryEscherichia coliCascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation
TheEscherichia coli typeI-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interferencein vivo. Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.