Plasmid and strain construction
Plasmids and strains used in this study are provided in Supplementary Tables S1 and S2, respectively. Plasmids encoding unit-sized crRNA were constructed by cloning synthetic dsDNA duplex fragments (gBlocks from IDT Inc.) containing T7 A1 or trp promoter and crRNA coding sequences followed by (T)8 track into EcoNI and KpnI sites of pACYCDuet-1 vector (Novagen). Three T7 A1 promoter containing plasmids were prepared: pG8crRNA contains g8 spacer sequence targeting M13 phage DNA (genome positions 1358–1389); the plasmid pSpT5_crRNA contains T5 spacer sequence targeting T5 phage DNA (genome positions 1869–1900, complementary strand); the plasmid pSpλ_crRNA contains λ spacer sequence targeting phage λ DNA (genome positions 29798–29829, complementary strand). Spacer sequences were chosen based on the presence of functional PAM sequences upstream of corresponding protospacers in phage genomes. In plasmid pG8trp_crRNA, the g8 crRNA coding sequence was cloned under control of the _E. coli trp operon promoter.
Mutation C1T at the first position of the spacer was introduced into pG8_crRNA by QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer's protocol, creating plasmid pG8_crRNA_C1T. Plasmids encoding Cascade subunits and CRISPR cassette containing multiple g8 spacers are described in (5,8–9,23). The pT7Blue-based plasmids carrying a 209-bp M13 fragment with the g8 protospacer (genome positions 1311–1519) with or without an escape mutation C1T at the first position of the protospacer is described in (18).
Strains producing Cascade proteins for in vitro experiments are described in (18,23). Strains KD390, KD477 and KD599 created for this work are derivatives of KD263 (18) and were constructed using the previously published Red recombinase protocol (24).
RNA extraction and Northern blotting
The procedure was performed essentially as described (25). Total RNA was isolated from 2 ml of E. coli cells grown until OD600 reached 1 and lysed by 5-min treatment using Max Bacterial Enhancement Reagent with subsequent purification by the TRIzol reagent (Invitrogen). RNAs bound to Cascade were extracted from purified complexes by TRIzol reagent. 10 μg of total RNAs or 20–40 ng of Cascade-purified RNAs were separated on a denaturing 8 M urea - 12% polyacrylamide gel and electrophoretically transferred to Hybond-XL membrane (GE Healthcare) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The membrane was dried and then UV cross-linked. ExpHyb hybridization solution (Clontech) was used for hybridization according to manufacturer's instructions for 1 h at 37°C with 32P-end labeled g8-spacer specific probe (5′-GCGGGATCGTCACCCTCAGCAGCGAAAGACAG-3′).
Cascade or Cascade subcomplexes lacking Cse1, Cas6e, or Cas6e and Cse1 were prepared from cells co-expressing appropriate cas genes (with or without a source of crRNA and were one-step affinity-purified on Strep-Tactin® column (IBA) using the N-terminal Strep-tag attached to the Cse2 subunit (23) according to manufacturer's protocol. N-terminal 6His-tagged Cse1 protein was IMAC purified from E. coli KD418 strain and further purified using 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. We also purified two Cascade forms (with and without Cas6e) from cells lacking any source of crRNA using a protocol published earlier (23).
CRISPR interference and adaptation assays
E. coli strain carrying a genomic CRISPR array with g8 spacer (KD263, (18)) or strains KD390, KD477 and KD599 which harbor only a CRISPR repeat in their genome and which were supplemented with plasmids encoding unit-sized crRNA were used to determine cell sensitivity to phage infection by a spot test method as described (8). To perform crRNA expression from the trp promoter, KD390 cells transformed with the plasmid pG8trpcrRNA were grown in minimal medium M9 supplemented with 0.4% glycerol and 34 μg/ml chloramphenicol at 37°C until OD660 nm reached 0.5, then 3-β-indoleacrylic acid (IAA) was added to the final concentration 10 μg/ml for crRNA expression; IPTG and arabinose were added to 1 mM each to induce _cas genes expression. After 4 h induction the cells were used for plating and spot test with the M13 phage. 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 host-phage variant, plaquing efficiency was determined in at least three independent experiments. Escape phage plaques were analyzed by sequencing through the g8 protospacer region as described in (8). For plasmid transformation efficiency assays, competent pre-induced cells were prepared and electroporated with 10 ng of DNA from protospacer carrying plasmid (pG8, pG8_mut or pT7blue as a control). Transformation efficiency was determined as ampicillin-resistant colony numbers per μg DNA. The experiments were repeated at least three times.
To monitor CRISPR adaptation, KD263 cells and cells (KD390, KD477, KD599) carrying the pG8_crRNA plasmid were transformed with protospacer carrying plasmid pG8, individual colonies were selected, and grown overnight at 37°C in LB broth supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol where needed. Aliquots of cultures were diluted 200-fold with fresh LB broth without ampicillin and supplemented with IPTG and arabinose to the final concentration 1 mM each. The cultures were grown at 37°C overnight. To monitor spacer acquisition, 1 μl of culture was added to 20 μl PCR reaction and amplification was performed 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.
In vitro transcription
Double-stranded DNA fragment containing unit-sized g8 crRNA sequence flanked upstream with the T7 A1 promoter and downstream with (T)8 track was prepared by PCR amplification from pG8crRNA plasmid. The resulting fragment had coordinates −96/+149 with respect to the transcription start site located at +1. To allow open complex formation, 3.3 nM transcription template was incubated with 100 nM of _E. coli σ70 RNA polymerase holoenzyme in 10 μl of transcription buffer containing 35 mM Tris-HCl, 70 mM NaCl, 7 mM MgCl2, 0.7 mg/ml BSA, (pH 7.9 at 25°C) for 10 min at 37°C. Transcription was initiated by the addition of NTPs (200 μM ATP and GTP, 100 μM CTP, and 10 μM of UTP). 5 μCi of α-32P-UTP (3000 Ci/mmol) was also added. After 5-min incubation at 37°C, reaction was terminated by the addition of equal volume of formamide. RNA products were separated by 20% polyacrylamide gel electrophoresis (PAGE) at denaturing condition (8 M urea) and analyzed by autoradiography.
The target g8 dsDNA (209 bp) was PCR amplified from pG8 plasmid containing g8 protospacer (Supplementary Table S1). The 5′ ends of the target ds-DNA fragment were labeled with 32P using T4 polynucleotide kinase 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 polynucleotide kinase (New England Biolabs). 32P-labeled DNA fragments were purified on micro Bio-SpinTM chromatography columns packed with Bio-Gel P-6 (BIO-RAD). Oxidative modification by KMnO4 was performed with 5-nM-labeled target DNA, 0.2–0.4 μM of Cascade complex and, where necessary, 0.5–1 μM crRNA in 10 μl binding buffer (40 mM Tris-HCl, pH 8.0, 50 mM NaCl). When chemically synthesized crRNA was added, Cascade and crRNA were first mixed and incubated at 37°C for 5 min to allow Cascade-crRNA complex formation. Target DNA was added to Cascade-crRNA complex and incubated at 37°C for 25–30 min. The 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 10 μl of 1% β-mercaptoethanol and 5 μg of calf thymus DNA in 50 μl of 10 mM Tris-HCl (pH 8.5) and 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 μl of 10 mM Tris-HCl (pH 8.5) and supplemented with 15 μl of formamide loading buffer with Bromophenol Blue and Xylene Cyanol. Samples were separated by electrophoresis in 8% denaturing polyacrylmide gels containing urea. After electrophoresis, the gel was fixed in 10% acetic acid, dried and exposed to a phosphorus screen overnight. The images were scanned using PhosphorImager (Molecular Dynamics) and analyzed using ImageQuantMac v 1.2.
High throughput sequence data analysis
Raw sequencing data were analyzed using ShortRead and BioStrings (26) packages. Illumina-sequencing reads were filtered for quality scores of ≥20 and reads containing two repeats (with up to two mismatches) were selected. Reads that contained 33-bp sequences between two CRISPR repeats were next selected and considered as spacers. Spacers were next mapped on the pG8 plasmid with no mismatches allowed. R scripts were used for spacers statistics and Circos (27) was used for graphical representation of the data.
Article TitleThe Cas6e ribonuclease is not required for interference and adaptation by theE. colitype I-E CRISPR-Cas system
CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In theEscherichia colitype I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system inE. coliusing unit-sized crRNAs generated by transcription.