Materials and Methods

Determining the Specificity of Cascade Binding, Interference, and Primed AdaptationIn Vivoin theEscherichia coliType I-E CRISPR-Cas System

MATERIALS AND METHODSStrains and plasmids. All strains, plasmids, oligonucleotides, and purchased, chemically synthesized double-stranded DNA (dsDNA) fragments are listed in Table S5 in the supplemental material. All strains used were derivatives of MG1655 (59). CB386 has been previously described (38). CB386 contains a chloramphenicol resistance cassette in place of cas3. We removed this cassette using Flp recombinase, expressed from plasmid pCP20 (60), to generate strain AMD536. Epitope-tagged strains AMD543 and AMD554 (Cse1-FLAG3 and FLAG3-Cas5, respectively) are derivatives of CB386 and were generated using the previously described FRUIT method of recombineering (61). Cse1 was C-terminally tagged in AMD543 by inserting a FLAG3 tag immediately upstream of codon 495 using oligonucleotides JW6364 and JW6365. Tagging of Cse1 resulted in an 8-amino-acid C-terminal truncation. We predicted on the basis of phylogenetic comparisons and of structural data (49) that this truncation would not impact the function of Cse1. Cas5 was N-terminally tagged in AMD554 by inserting FLAG3 using oligonucleotides JW6272 and JW6273. LC060 is a derivative of AMD536 and was generated using (i) FRUIT (61) with oligonucleotides JW7537-JW7540 to delete the CRISPR-II locus, (ii) P1 transduction of the CB386 (Δcas3 Pcse1)::(Cat::PJ23199) region, (iii) FRUIT (61) to C-terminally tag Cse1 with FLAG3 (as described above for AMD543), and (iv) pCP20-expressed Flp recombinase (60) to remove the cat cassette. LC074 is a derivative of AMD536 in which the CRISPR-I array was deleted using FRUIT (61) with oligonucleotides JW7529 and JW7530 and a synthesized dsDNA fragment (gBlock 14148263; Integrated DNA Technologies, Inc.). LC077 is a derivative of LC074 in which Cse1 was C-terminally tagged with FLAG3 (as described above for AMD543). AMD566 is a derivative of AMD536 in which Cse1 was C-terminally tagged with FLAG3 (as described above for AMD543). LC099 is a derivative of AMD566 in which the off-target binding site for Cascade in yggX was mutated using FRUIT (61) with oligonucleotides JW7635 to JW7638. LC103 is a derivative of AMD536 in which the yggX gene was replaced with a kanamycin resistance cassette using P1 transduction from the Keio Collection ΔyggX::Kanr strain (62). LC106 is a derivative of LC103 with an unmarked, scar-free deletion of cas1 made using FRUIT with oligonucleotides JW7898 to JW7901. AMD688 is a strain that contains a previously reported yfp reporter construct that can be used to measure adaptation levels (43). AMD688 was constructed by P1 transduction of the Δcas3::cat cassette from CB386 into MLS1003 (provided by the Lundgren laboratory). The cat gene was removed using Flp recombinase, expressed from plasmid pCP20 (60). AMD688 has an intact copy of the CRISPR-I array (cotransduced with the Δcas3::cat cassette from CB386) but lacks the CRISPR-II array.TABLE S5 Strains, plasmids, oligonucleotides, and chemically synthesized dsDNA fragments used in this study. Download TABLE S5, PDF file, 0.2 MB.Copyright © 2018 Cooper et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Plasmids that express crRNAs targeting the lacZ promoter (pCB380) and araB promoters (pCB381) have been described previously (38). All other crRNA-expressing plasmids used were derivatives of pAMD179. pAMD179 was constructed by amplifying a DNA fragment from plasmid pAMD172 (Integrated DNA Technologies, Inc.) using oligonucleotides JW6421 and JW6513. This DNA fragment was cloned into pBAD24 (63) cut with NheI and HindIII (NEB) using the In-Fusion method (Clontech). The inserted fragment contains two repeats from the CRISPR-I array, separated by a stuffer fragment containing XhoI and SacII restriction sites, and an intrinsic transcription terminator downstream of the second repeat. To clone individual spacers, pairs of oligonucleotides were annealed, extended, and inserted using In-Fusion (Clontech) into the XhoI and SacII sites of pAMD179 to generate pLC008 (with oligonucleotides JW6518 and JW7911), pLC010 (with oligonucleotides JW6518 and JW7912), and pAMD189 (with oligonucleotides JW7598 and JW7693). Note that the derivatives of sp1.8 expressed from pLC008 and pLC010 differ from sp1.8 at the last two nucleotide positions to facilitate cloning. These mismatches are not expected to affect crRNA function (23, 38).pLC021, pLC022, and pLC057 are derivatives of pBAD24 (63) that contain a protospacer matching the off-target Cascade binding site in yggX (pLC021), a protospacer with a perfect match to sp1.8 (pLC022), or a protospacer with a perfect match to sp1.2 (pLC057). These plasmids were constructed by annealing and extending pairs of oligonucleotides (JW7913 and JW7914 for pLC021, JW7924 and JW7925 for pLC022, and JW9131 and JW9132 for pLC057) and cloning the resultant DNA fragments into the EcoRV and SphI sites of pBAD24. pAMD191 is a derivative of pBAD33 (63) that expresses cas3 under arabinose control. To construct pAMD191, cas3 was amplified by colony PCR using oligonucleotides JW7736 and JW7738. The PCR product was cloned into the SacI and HindIII sites of pBAD33 using In-Fusion (Clontech). All protospacers described in the Fig. 5 legend were cloned into plasmid pLC020, the “preprotospacer plasmid,” which is a derivative of pBAD24 (63). pLC020 was generated by cloning the ~500-bp region upstream of E. coli thyA (amplified by colony PCR using oligonucleotides JW8040 and JW8128) and the ~500-bp region downstream of E. coli thyA (amplified by colony PCR using oligonucleotides JW8042 and JW8043) into the EcoRI site of pBAD24 using In-Fusion (Clontech), simultaneously generating a new EcoRI site between the upstream and downstream regions of thyA. The thyA gene was then amplified by colony PCR using a universal forward primer (oligonucleotide JW8129) and each of 13 reverse primers (oligonucleotides JW8130, JW8139, JW8145, JW8169, JW8499 to JW8502, and JW8675 to JW8679) containing the 13 protospacer variants described in the Fig. 5 legend The resulting PCR products were cloned into the EcoRI site of the pBAD24 derivative using In-Fusion (Clontech) to generate plasmids pLC023 to pLC035 (see Table S5 for details). Note that plasmids pLC024 and pLC025 differ from pLC023 and from pLC026 to pLC035 at the nucleotide position immediately adjacent to the protospacer, on the PAM-distal side. Differences at this nucleotide position are not expected to affect Cascade binding, interference, or primed adaptation.ChIP-qPCR. Cells were grown overnight in LB and subcultured in LB supplemented with 0.2% arabinose and 100 µg/ml ampicillin at 37°C with aeration to an optical density at 600 nm (OD600) of ~0.6. AMD566 and LC099 were used with either pLC008 or pLC010 for ChIP-qPCR. ChIP-qPCR was performed as described previously (64), except that 2 µl anti-FLAG M2 monoclonal antibody (Sigma) and 1 µl anti-σ54 monoclonal antibody (NeoClone) were included and processed simultaneously in the immunoprecipitation step. qPCR was performed using oligonucleotides JW7490 to JW7491 (amplifying the off-target site in yggX) and JW7922 to JW7923 (amplifying the region upstream of hypA). Since σ54 is known not to bind within yggX (65), we were able to normalize binding of Cse1 within yggX to the binding of σ54 upstream of hypA.ChIP-seq. Strains AMD543, LC060, LC077, AMD543 and AMD554 with pCB380 and pCB381, and LC077 were used for ChIP-seq analysis of Cse1-FLAG3 and FLAG3-Cas5, except that ampicillin was included only for the experiments involving a crRNA-expressing plasmid and arabinose was included only for the experiments using pLC008. Cells were grown and processed as described for ChIP-qPCR. ChIP-seq was performed in duplicate, following a previously described protocol (66) using 2 µl anti-FLAG M2 monoclonal antibody (Sigma). Sequencing was performed on an Illumina High-Seq 2000 instrument (Next-Generation Sequencing and Expression Analysis Core, State University of New York at Buffalo) or an Illumina Next-Seq instrument (Wadsworth Center Applied Genomic Technologies Core). ChIP-seq data analysis was performed as previously described (67), with reads mapped to the updated MG1655 E. coli genome (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"U00096.3","term_id":"545778205","term_text":"U00096.3"}}U00096.3). Relative sequence coverage values were calculated by calculating the sequence read coverage at a given genomic location as follows: total number of sequence reads in the run/100,000. Values plotted in Fig. 1A and ​andBB and ​and2A2A and ​andDD represent the maximum values in 1-kbp regions across the genome. R2 values comparing ChIP-seq data sets were calculated by comparing levels of read coverage at peak centers for all peaks identified for the analyzed data sets. Read coverage at peak centers was determined using a custom Python script. Sequence motifs were identified using MEME (version 4.12.0) (68) with default parameters.RNA-seq. RNA-seq was performed in duplicate with strains AMD536 and LC074, with and without pAMD191. Cells were grown overnight in LB and subcultured in LB (supplemented with 0.2% arabinose and 100 µg/ml ampicillin for experiments involving pAMD191) at 37°C with aeration to an OD600 of ~0.6. RNA was purified using a modified hot-phenol method, as previously described (69). Purified RNA was treated with 2 µl DNase (Turbo DNA-free kit; Life Technologies, Inc.) for 45 min at 37°C, followed by phenol extraction and ethanol precipitation. A Ribo-Zero kit (Epicentre) was used to remove rRNA, and strand-specific cDNA libraries were created using a ScriptSeq 2.0 kit (Epicentre). Sequencing was performed using an Illumina Next-Seq instrument (Wadsworth Center Applied Genomic Technologies Core). Differential RNA expression analysis was performed using Rockhopper (version 2.03) with default parameters (70). Differences in RNA levels were considered statistically significant for genes with false-discovery-rate (q) values of ≤0.01.Plasmid transformation efficiency assay. LC103 was transformed with either empty pBAD33 or pAMD191 (expresses cas3), and these strains were then transformed with pBAD24 (no protospacer) or pLC021 (protospacer with a perfect match to sp1.8) or pLC022 (protospacer with an imperfect match to sp1.8, corresponding to the off-target site in yggX) or pLC057 (protospacer with a perfect match to sp1.2). Cells were plated on M9 medium supplemented with 0.2% glycerol, 0.2% arabinose, 100 µg/ml ampicillin, and 30 µg/ml chloramphenicol at 37°C. After overnight growth, colonies were counted, and the relative levels of transformation efficiency were calculated as ratios of transformants for pAMD191-containing cells to transformants for pBAD33-containing cells for each transformed protospacer-containing plasmid.PCR to assess primed adaptation. Primed adaptation was assessed for AMD536 with pAMD191 and either pLC021 or pLC022 (Fig. 4B) and for MG1655, AMD536, AMD543, and AMD544 with pAMD191 and pAMD189 (expresses a self-targeting crRNA; see Fig. S1 in the supplemental material). Cells were grown overnight in LB supplemented with 100 µg/ml ampicillin and 30 µg/ml chloramphenicol at 37°C with aeration and were subcultured the next day in LB supplemented with chloramphenicol and 0.2% arabinose at 37°C with aeration for 6 h. Cells were pelleted from 1 ml of culture by centrifugation, and cell pellets were frozen at −20°C. PCRs were then performed on the cell pellets, amplifying the CRISPR-II array using oligonucleotides JW7818 and JW7819. PCR products were visualized on acrylamide gels.Sequence analysis of protospacers from a pooled ChIP library. LC099 was grown with each of the 13 protospacer variant plasmids (pLC23 to pLC035) overnight in LB supplemented with 100 µg/ml ampicillin. Ten-milliliter subcultures were grown in LB supplemented with 100 µg/ml ampicillin and 0.2% arabinose at 37°C with aeration to an OD600 of ~0.6. Three-milliliter volumes from all cultures were combined. ChIP was performed on mixed cultures using 2 µl M2 anti-FLAG monoclonal antibody (Sigma), as previously described (64). A Zymo PCR Clean and Concentrate kit was used to purify ChIP and input DNA. A 50-µl FailSafe (Epicentre) PCR using FailSafe PCR 2× PreMix C and 5.48 ng of ChIP DNA was performed following the manufacturer’s instructions, using oligonucleotide JW8567 and each of oligonucleotides JW8537, JW8556, JW8557, JW8558, JW8559, JW8561, JW8562, JW8563, JW8564, and JW8565 (these incorporate different Illumina indexes). PCR products were purified and concentrated using 0.8× AMPure beads (Beckman Coulter, Inc.; Life Sciences) and sequenced on an Illumina Mi-Seq instrument (Wadsworth Center Applied Genomic Technologies Core). Sequence reads were mapped to each of the 13 protospacer variants using a custom Python script. Relative levels of protospacer abundance in input and ChIP samples for each protospacer were normalized to the total sequence reads. Values for normalized protospacer abundance were further normalized to values from the input sample. Protospacer abundance values are reported relative to those for the optimal protospacer (variant I in Fig. 5).Measuring interference for a pooled protospacer library. Overnight cultures of LC106 strains with each of the 13 protospacer plasmids (pLC023 to pLC035) were grown in LB with 100 µg/ml ampicillin and 30 µg/ml kanamycin. All 13 cultures were combined to make a single subculture (7.7 µl of each overnight culture into a single 10-ml culture). Electrocompetent cells were made and transformed with either empty pBAD33 or pAMD191 (pBAD33-cas3). Transformants were plated onto M9 agar supplemented with 0.2% glycerol, 0.2% arabinose, and 30 µg/ml chloramphenicol and were grown overnight at 37°C. Cells were scraped off plates and washed in LB, and protospacers were PCR amplified from cell pellets with oligonucleotide JW8567 and each of oligonucleotides JW8537, JW8558, JW8559, JW8562, JW8563, and JW8566 (these incorporate different Illumina indexes). PCR products were purified and concentrated with 0.8× AMPure beads (Beckman Coulter, Inc.; Life Sciences) and sequenced using an Illumina MiSeq instrument (Wadsworth Center Applied Genomic Technologies Core). Sequence reads were mapped to each of the 13 protospacer variants using a custom Python script. Relative interference efficiency levels were calculated for each protospacer variant by dividing the number of sequence reads from cells transformed with empty pBAD33 by the number of sequence reads from cells transformed with pAMD191 (pBAD33-cas3) and normalizing to the value for the protospacer with a CCG PAM (variant ii in Fig. 5).Measuring primed adaptation using a yellow fluorescent protein (YFP) fluorescent reporter. MLS1003 was transformed with each of plasmids LC023 to LC035, and each of the resulting strains was transformed with cas3-expressing plasmid pAMD191. Cells were grown overnight at 37°C with shaking in LB supplemented with 100 µg/ml ampicillin and 30 µg/ml chloramphenicol. Cells were subcultured 1:100 for 6 h in LB supplemented with 0.2% l-arabinose and 20 µg/ml chloramphenicol at 37°C with shaking. Cells were pelleted by centrifugation and resuspended in M9 minimal medium in twice the original volume (OD600 values of ~1). Cells were transferred to 5-ml polystyrene round-bottom tubes and were analyzed by flow cytometry for single-cell detection of yfp expression using a BD FACSAria IIU cell sorter. A total of 100,000 events were recorded for each sample. Experiments were performed for between 3 and 10 independent biological replicates.Accession numbers. All next-generation sequencing data sets described in this paper are available at EBI ArrayExpress with the accession numbers E-MTAB-5970, E-MTAB-5971, E-MTAB-6446, E-MTAB-5972, and E-MTAB-5969.

Article TitleDetermining the Specificity of Cascade Binding, Interference, and Primed AdaptationIn Vivoin theEscherichia coliType I-E CRISPR-Cas System

Abstract

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptationin vivo, for the type I-E system ofEscherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenousE. colicrRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.


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