Materials and Methods

Identification of Spacer and Protospacer Sequence Requirements in theVibrio choleraeType I-E CRISPR/Cas System

MATERIALS AND METHODSBacterial strains and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 3. Bacteria were cultivated at 37°C in Luria Broth (LB) agar or in LB. The medium was supplemented with ampicillin (Amp; 50 μg/ml), kanamycin (Kan; 50 μg/ml), spectinomycin (Spec; 100 μg/ml), and/or streptomycin (Sm; 100 μg/ml) when appropriate. For induction, LB agar plates were supplemented with 100 μM isopropyl-β-d-thiogalactopyranoside (IPTG).TABLE 3Bacterial strains and plasmids used in this studyStrain or plasmidDescriptionSource or referenceV. cholerae strains    E7946El Tor biotype, serogroup O1; Smr52    KS916E7946 with CRISPR/Cas array from O395 under the control of the tac promoter; Kanr ΔlacZ::Spec15    AC6625KS916, wild-type lacZ by natural transformation of lacZ locus from E7946; KanrThis studyPlasmids    pCRISPRExpression plasmid containing the V. cholerae CRISPR spacer array under the control of the tac promoter in the pMMB67EH background; Ampr15    pDL1301Conjugation plasmid with p15a minimal origin, RP4 oriT, and spectinomycin adenyltransferase gene aad9; SpecrThis studyOpen in a separate windowBioinformatic analysis of CRISPR repeats in deposited sequences of V. cholerae. Additional strains containing the 28-bp V. cholerae O395 CRISPR repeat (5′-GTCTTCCCCACGCAGGTGGGGGTGTTTC-3′) were identified using BLAST to search the NCBI whole-genome shotgun contigs database, restricting results to the family Vibrionaceae. This method would identify O395 CRISPR repeats when present in other Vibrio species; however, we found that only V. cholerae strains contained the perfect repeat. Extraction of spacer content and protospacer mining were automated using a custom Python script (https://github.com/camillilab/spacer_miner). Briefly, identified contigs containing repeat sequences were retrieved from NCBI. Spacers were extracted if they had sequences no longer than 50 nucleotides, were flanked by perfect repeat sequences, and did not contain ambiguous nucleotides. Each unique spacer was then compared with BLAST to the NCBI nonredundant nucleotide database in order to identify putative protospacers. Putative protospacers were considered if there was at least 93% identity to the corresponding spacer over 96% of the spacer sequence, which permits up to two mismatches and one missing or additional base in the protospacer hit. Sequence logos were generated using unique spacers and nucleotides 3′ of identified protospacers using WebLogo (51).Conjugation assays. The donor plasmid pDL1301, an RP4-conjugatable plasmid that confers resistance to spectinomycin, and its variants were transferred using E. coli SM10λpir. Donor and recipient AC6625 strains were grown to an optical density at 600 nm ( OD600) of 1.0. Then 500 μl of the donor and 500 μl of the recipient were pelleted, washed once in phosphate-buffered saline (PBS), and resuspended in 50 μl PBS. A 1:1 mixture was applied to a sterile filter (pore size, 0.22 μm; Millipore) on an LB plate and was incubated at 37°C for 2 h. Bacteria were recovered from the filter by vortexing in 500 μl PBS. Serial dilutions were plated onto a medium selective for recipient V. cholerae and exconjugates and were plated separately in the presence of IPTG. The conjugation efficiency was calculated as the number of exconjugates divided by the total number of viable V. cholerae recipients.Construction of targeting plasmids. CRISPR targeting plasmids were generated using the previously constructed pCRISPR backbone (15). New spacers were constructed using annealed and phosphorylated oligonucleotides that included the targeting region flanked by appropriate 5′ and 3′ overhangs to facilitate ligation into pCRISPR. The resulting double-stranded DNAs were cloned into pCRISPR by Golden Gate cloning using BsaI-HF (New England Biolabs). Ligation products were purified and used to transform electrocompetent E. coli SM10λpir. DNA from individual clones was isolated, and the targeting sequence was verified by Sanger sequencing. These plasmids were then mated into the V. cholerae targeting strain AC6625.Generation of mutant donor and targeting plasmids. The target aad9 gene in donor plasmid pDL1301 was modified in eight independent sites by site-directed PCR mutagenesis to yield eight new donor plasmids possessing one silent mutation each (aad9). The location of each of these mutations is listed in Table 2. Eight new spacers that target these mutated sites were introduced into targeting plasmid pCRISPR as previously described to yield eight new targeting plasmids (crRNA). pCRISPR constructs pyrimidine-1 and pyrimidine-3 and pDL1301 constructs aad9A153G and aad9A456G were constructed in the same manner.

Article TitleIdentification of Spacer and Protospacer Sequence Requirements in theVibrio choleraeType I-E CRISPR/Cas System

Abstract

Additional strains containing the 28-bpV. choleraeO395 CRISPR repeat (5′-GTCTTCCCCACGCAGGTGGGGGTGTTTC-3′) were identified using BLAST to search the NCBI whole-genome shotgun contigs database, restricting results to the familyVibrionaceae. This method would identify O395 CRISPR repeats when present in otherVibriospecies; however, we found that onlyV. choleraestrains contained the perfect repeat. Extraction of spacer content and protospacer mining were automated using a custom Python script (https://github.com/camillilab/spacer_miner). Briefly, identified contigs containing repeat sequences were retrieved from NCBI. Spacers were extracted if they had sequences no longer than 50 nucleotides, were flanked by perfect repeat sequences, and did not contain ambiguous nucleotides. Each unique spacer was then compared with BLAST to the NCBI nonredundant nucleotide database in order to identify putative protospacers. Putative protospacers were considered if there was at least 93% identity to the corresponding spacer over 96% of the spacer sequence, which permits up to two mismatches and one missing or additional base in the protospacer hit. Sequence logos were generated using unique spacers and nucleotides 3′ of identified protospacers using WebLogo (51).


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