Materials and Methods

CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system

MATERIALS AND METHODSPlasmid constructionThe leader sequence and two repeat–spacer units of the CRISPR array was PCR-amplified from the S. thermophilus genome and cloned into the pWAR228 backbone plasmid by overlap PCR to generate pCRISPR. Leader sequence mutations were generated via inverse PCR and ligation of linearized plasmid using pCRISPR as the template. All plasmid constructs were verified by DNA sequencing and are listed in Supplemental Table S1.Protein purificationThe cas1, csn2 and cas9 genes were amplified by PCR from the S. thermophilus genome and cloned into pET expression vectors to generate 6x-histidine-tagged proteins at the C-terminus (pET21d;Cas1 and Cas9) or N-terminus (pET24d; Csn2). The cas2 gene was subcloned into pBAT4 expression vector to generate 6x-histidine-tagged SUMO Cas2 proteins at the N-terminus (pSAT1 and pSENP kindly provided by Dr Scott Bailey, Johns Hopkins University). Expression vectors were transformed into Escherichia coli BL21-Star cells (DE3, Stratagene). Cells were grown at 37°C in 1 L cultures of Luria broth to an OD600 of 0.6, and protein expression was induced overnight at room temperature by the addition of ispopropylthio-β-d-galactoside (IPTG) to a final concentration of 1 mM. The cells were pelleted, resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 10% glycerol, 20 mM imidazole and 5 mM 2-mercaptoethanol (BME), pH 7.5) and disrupted by sonication (Misonix Sonicator 3000). The lysate was cleared by centrifugation at 3500 rpm for 20 min at 4°C and His-tagged proteins were purified by Ni2+ affinity column chromatography (1.5 ml of HisPur Ni-NTA Resin (Thermo Scientific)) using a stepwise increase of imidazole (20, 50, 100 and 500 mM). The protein samples were dialyzed at 4°C in dialysis buffer (20 mM Tris, 150 mM KCl, 10% glycerol, 5 mM 2-mercaptoethanol (BME), pH 7.5) prior to performing activity assays. Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining (Supplemental Figure S2).Generation of DNA substratesDNA oligonucleotides were from Eurofins MWG Operon with the exception of hairpin DNA substrates used in Figure ​Figure4,4, which were from Integrated DNA Technologies and the sequences are given in Supplemental Table S2. Oligonucleotides were annealed by an incubation temperature gradient for 1 min at 95°C decreasing by 1°C each minute, down to 23°C. Annealed double-stranded substrates were run on a non-denaturing 15% polyacrylamide gel containing 1× TBE (89 mM Tris base, 89 mM Boric acid, 2 mM EDTA, pH 8.0), followed by ethidium bromide post-staining to verify proper annealing prior to radiolabeling. The annealed DNA substrates used as pre-spacers were 5′ end-labeled with T4 polynucleotide kinase (New England Biolabs (NEB)) in a 20 μl reaction containing 20 pmol oligonucleotide, 150 μCi of γ-32P ATP (6000 Ci/mmol; Perkin Elmer), 1× T4 PNK buffer and 10 U of T4 kinase (NEB).Open in a separate windowFigure 4.Cas1–Cas2 spacer integration reaction is directional. (A) Detection of full-site and half-site integration products with spacer hairpin CRISPR targets (top panel) and leader hairpin CRISPR targets (bottom panel) with unmodified (OH/OH) and modified (OH/dd, dd/OH, dd/dd) pre-spacers show site of the first transesterification reaction. (B) Time-course of spacer integration assay using unmodified pre-spacer and spacer hairpin CRISPR target. Quantification of B (bottom panel).Integration assay with radiolabeled pre-spacerFor plasmid integration assays, individually purified recombinant Cas1 and Cas2 proteins at 2.5 μM each were added to a reaction containing 5 nM plasmid DNA, 20 nM 5′γ-32P ATP-radiolabeled DNA pre-spacer substrate, and integration buffer (20 mM Tris (pH 7.5), 100 mM KCl, 10 mM MnCl2, 5 mM 2-mercaptoethanol). This reaction was incubated at 37°C for 1 h and then quenched by the addition of 1 μg Proteinase K (ThermoFisher Scientific), 0.5% SDS, 1 mM EDTA and incubated at 50°C for 30 min. The products were analyzed on a 0.8% agarose gel pre-stained with ethidium bromide. After gel electrophoresis, the gels were dried on blot absorbent filter paper (Bio-Rad) overnight at room temperature using a vacuum gel dryer (Bio-Rad, Model 583 Gel Dryer). Radioactivity was detected with a phosphorimager (Storm 840 Scanner GE Healthcare).For linear DNA target integration assays, individually purified recombinant Cas1 and Cas2 proteins both at 250 nM were added to a reaction containing 100 nM DNA CRISPR target, and integration buffer (described above). This reaction was incubated at 25°C for 5 min and then 20 nM 5′γ-32P ATP-radiolabeled DNA pre-spacer substrates were added and 10 μl samples were removed at 15 sec, 1 min and 15 min or incubated at 25°C for 1 hour. Reactions were quenched by the addition of equal volume (10 μl) of 95% formamide and 50 uM EDTA and incubated at 98°C for 5 min and separated on a 12% (8.0 M urea) denaturing polyacrylamide gel. Radiolabeled Decade Markers (Life Technologies) were used to determine the size of observed products. After gel electrophoresis, the gels were dried for 1 h at 90°C (Bio-Rad, Model 583 Gel Dryer) and radioactivity was detected by phosphorimaging as described above.Repeat mutation adaptation assay in vivoFor in vivo integration assays, pCas1/Cas2/Csn2/Cas9 with a minimal CRISPR array (pCRISPR) was used as template as previously described (41) and inverse PCR was used to introduce both insertions and deletions of the repeat sequence. Plasmid constructions were verified by sequencing and transformed into S. thermophilus DGCC7710 strain via electroporation (59). S. thermophilus harboring the plasmids were grown in LM17 liquid medium supplemented with 2 μg/mL chloramphenicol for 16 hours. Cells from each strain were harvested, pelleted and genomic DNA was extracted using the Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine CA) and used as PCR template. Primers matching the leader and plasmid sequence were used for PCR amplification of the CRISPR array on the plasmid. PCR products were run on 2.5% TAE-agarose gels, pre-stained with ethidium bromide to assess CRISPR array expansion. Bands representing an expanded CRISPR array were gel excised using the Zymo Gel Extraction DNA Recovery Kit (Zymo Research, Irvine, CA, USA), purified and sequenced by high-throughput sequencing. Plasmid constructs are listed in Supplemental Table S1.Pre-spacer integration high-throughput sequencingLibrary preparation To sequence integration events, the spacer integration assay was performed as described above using unlabeled pre-spacer. After incubation, DNA was isolated using the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA). For the plasmid integration samples, excess un-integrated pre-spacer was removed using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN). Illumina adapter sequence with an N10 random primer was annealed to the plasmid DNA and extended (thermocycler conditions: 98°C for 30 s, 25°C for 30 s, 35°C for 30 s, 45°C for 30 s and 72°C for 5 min). Excess adapter was then removed using AMPure beads, and PCR was performed to amplify plasmid DNA that contained integrated pre-spacer: forward primers were specific for the pre-spacer, while reverse primers targeted the Illumina adapter introduced with the random anneal and extension step. The resulting amplicons captured both full-site and half-site integration events with no apparent discrimination. Illumina barcodes and adapter sequences were added with a final PCR and the resulting library was separated on a 1% agarose gel. DNA in a 400–700 bp size range was selected and isolated using the Zymo Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA). Sequencing was performed on an Illumina MiSeq with a 100 × 50 cycle run. Only the 100 bp Read 1 data was used in this analysis.For the minimal linear CRISPR substrate products, 1 μl of eluted DNA was used as a PCR template. Primers to add Illumina adaptor sequences were annealed to the newly integrated spacer and the 3′ end of either the plus or minus strand of the CRISPR substrate. DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA) was used to isolate the PCR product, and 1 μl of this product was used as the template for a second PCR using primers to add Illumina barcodes. These products were purified on a 1% agarose gel and extracted with a Gel Purification Kit (Zymo Research, Irvine, CA, USA).Mapping integration events After sequencing, samples were de-multiplexed by barcode and analyzed to determine sites of integration. For plasmid data, the complete pre-spacer sequence was located in each read and 50 bp of sequence immediately downstream from the end of the pre-spacer was extracted and aligned to the appropriate plasmid reference using Bowtie (62). To visualize the distribution of integration events, alignment output files were converted into coverage files using bedtools (63) and displayed on a custom UCSC genome browser track hub (https://www.genome.ucsc.edu). To determine sequence preferences at the sites of integration, the base at the integration point, along with upstream and downstream context sequence, was extracted from the reference sequence with bedtools and used to make sequence logos (64). For the minimal linear CRISPR integration data, the spacer-target junction was determined from each read and counts for each potential integration point were totaled. Integration events are displayed as the percent of total reads for each position along the CRISPR target.Characterizing in vivo spacer integration into pCRISPR with repeat mutations Size selected and purified array amplicon libraries were sequenced on an Illumina MiSeq with a 250 × 50 cycle run (250 bp Read 1 data used in this study). Samples were de-multiplexed by barcode and then analyzed with custom python scripts to determine how new spacers were integrated. Briefly, the leader-repeat junction and the beginning of the second repeat were located in each read. The beginning of the second repeat was defined as the 3′ end of a set of hypothetical spacers, which ranged in size from 27 to 33 bp. This size range captures 99.9% of new type II-A spacers observed in spacer uptake assays with wildtype S. thermophilus. Each of the seven hypothetical spacers was aligned to a reference sequence including the genome and plasmid sequences using bowtie (62). Alignment outputs were then examined to determine the longest hypothetical spacer that aligned with no mismatches. This hypothetical spacer was considered the ‘true’ new spacer and its length was used to locate the position of the repeat–spacer junction, thereby allowing us to identify the integration site for each read. The number of reads supporting integration at each position along the pCRISPR array was counted and summarized and events are displayed as the percent of total reads for each position along the pCRISPR array.

Article TitleCRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR–Cas system

Abstract

Sequence data were deposited in the NCBI Sequence Read Archive under the BioProject ID PRJNA548779.


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