Materials and Methods

Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems

MATERIALS AND METHODSStrains and plasmid construction. See Table S2 in the supplemental material for a list of all strains used in this work. E. coli K-12 strain BW25113-T7 was generated by transferring araB::T7-RNAp-tetA from IY5163 to BW25113 by P1 transduction. Successful transduction was verified by PCR. BW25113-T7m2,5,7′ (Fig. 2A) was generated using three rounds of oligonucleotide-mediated recombination with ftsA-m257-spacer.recomb and the pKD46 plasmid encoding the λ red recombination genes (49, 50). The oligonucleotide contained two phosphorothioate linkages at each end to improve the recombination efficiency (51, 52). Successful recombinants were verified by PCR and by sequencing.See Table S2 in the supplemental material for a list of all plasmids used in this work. The origins of replication for the pCas3, pCasA-E, and pCRISPR plasmids used with E. coli and S. enterica belong to different incompatibility groups (26, 53). To generate the pCasA-E plasmid lacking the casABCDE operon (pCasA-E′), pCasA-E was digested with NcoI/NotI, blunt ended using Pfu polymerase, and ligated.To generate the pCRISPR plasmid, the pBAD18 plasmid (53) was linearized with XbaI and amplified by PCR using primers pBAD18.fwd/pBAD18.rev. A chemically synthesized gBlock (IDT) was then inserted downstream of the PBAD promoter by Gibson assembly (54). The gBlock encoded four repeats and three intervening spacers from the endogenous CRISPR locus in E. coli K-12 MG1655 (see Table S2 in the supplemental material), where the first spacer was modified to include a KpnI restriction site and an XhoI restriction site. These restriction sites allow the sequential insertion of engineered repeat-spacer pairs (see Fig. S2). Each pair was chemically synthesized as two oligonucleotides (IDT), phosphorylated with polynucleotide kinase, annealed, and ligated into the pCRISPR plasmid digested with KpnI and XhoI.The pBAD18-asd,msbA,ftsA,nusB plasmid was constructed in a manner similar to that for the pCRISPR plasmid, wherein a chemically synthesized gBlock (IDT) was inserted downstream of the PBAD promoter of the linearized pBAD18 plasmid by Gibson assembly (54). The gBlock encoded the first repeat-spacer sequence from the endogenous E. coli CRISPR locus, followed by five repeats and four intervening spacers targeting four different locations in E. coli Bw25113 (asd, msbA, ftsA, and nusB) (see Table S2 in the supplemental material).To generate pORI28 (55) with engineered spacers, pORI28 and each insert generated through PCR assembly were digested with BamHI and SacI and ligated together. To generate the insert encoding the lacZ1 spacer, template-free PCR was conducted with C1-lacZ1.fwd/C1-lacZ1.rev, followed by using the resulting product in a subsequent PCR with C1-BamHI.fwd/C1C3-SacI.rev. To generate the inserts encoding the lacZ2 and lacZ3 spacers, first the CRISPR3 leader region was amplified by PCR from LMD-9 genomic DNA with C3-leader.fwd/C3-leader.rev. Next, the resulting product was used as the template in a subsequent PCR with C3-leader.fwd/C3-lacZ2.rev or C3-leader.fwd/C3-lacZ3.rev. Finally, each PCR product was used as the template in a final round of PCR with C3-leader.fwd/C1C3-SacI.rev. All oligonucleotides and enzymes were purchased from IDT and NEB, respectively. All cloned plasmids were verified by sequencing.Growth conditions. All E. coli and Salmonella strains were cultured in LB medium (10 g/liter tryptone, 5 g/liter yeast extract, and 10 g/liter sodium chloride) at 37°C and 250 rpm with appropriate antibiotics. The same strains were plated on LB agar (LB medium with 1.5% agar) supplemented with appropriate inducers and incubated at 37°C. S. thermophilus LMD-9 was cultured in Elliker broth (Elliker medium Difco supplemented with 1% beef extract) and plated on Elliker agar (Elliker broth with 1.5% agar) (56). Both culturing and plating of LMD-9 were conducted at 37°C. Antibiotics were administered at the following final concentrations: 50 µg/ml streptomycin, 50 µg/ml kanamycin, 50 µg/ml ampicillin, 2 µg/ml chloramphenicol, and 2 µg/ml erythromycin. Inducers were administered at the following final concentrations: 0.1 mM IPTG and 0.02% l-arabinose.Design of native CRISPR RNAs. An overview of the approach to design and insert spacer sequences into the CRISPR array within the pCRISPR plasmid is shown in Fig. S2 in the supplemental material. The spacers were designed by identifying one of the known PAMs for the type I-E CRISPR-Cas system in E. coli (AAG, GAG, GAG, and ATG). The downstream 32 nucleotides (nt) were then used as the spacer within the engineered repeat-spacer pair. Note that the last two nucleotides of the spacer are fixed as TC because of the adopted cloning strategy (see Fig. S2). However, these nucleotides fall well outside the seed region and therefore are expected to have a negligible effect on targeting.The spacers for S. thermophilus were designed by identifying a known PAM for CRISPR1 (NNAGAAW) or for CRISPR3 (NGGNG) (10). The sequence of the 31 nt upstream of each PAM was integrated into oligonucleotides that were used to generate a leader region followed by a single repeat-spacer-repeat that was subsequently cloned into pORI28. This construct relies on processing through the native tracrRNA and RNase III.Transformation assay. Freezer stocks of E. coli and Salmonella strains harboring pCas3 and pCasA-E (or pCasA-E′) were streaked to isolation on LB agar. Individual colonies were inoculated into 3 ml of LB medium and shaken overnight at 37°C. The cultures then were back diluted into 25 ml of LB medium and grown to an A600 of 0.6 to 0.8, which was measured on a Nanodrop 2000c spectrophotometer (Thermo Scientific). The cells were then pelleted and washed with ice-cold 10% glycerol 2 times before being resuspended in 150 to 350 µl of 10% glycerol. The resuspended cells (50 µl) were transformed with 50 ng of pCRISPR or pCRISPR encoding the indicated spacer, using a MicroPulser electroporator (Bio-Rad), and recovered in 300 µl of SOC medium (Quality Biological) for 1 h (E. coli) or for 2 h (Salmonella). After the recovery period, 200 µl of different dilutions of the cells were plated on LB agar with inducers. The transformation efficiency was calculated by dividing the number of transformants for the tested plasmid by the number of transformants for the original pCRISPR plasmid. To normalize for experimental variability in transformation efficiency, the same batch of cells prepared for electroporation was transformed with each tested plasmid and the original pCRISPR plasmid.S. thermophilus strain LMD-9 harboring pTRK669 was grown in 50 ml of Elliker broth and prepared for electroporation as described previously, which concentrated the culture 100-fold (57). The resuspended cells (50 µl) were transformed with 1 µg of the pORI28 control plasmid or pORI28 containing the indicated spacer. Transformed cells were recovered in 950 µl of Elliker broth overnight and plated on Elliker agar. Plates were then incubated for 48 h in a Coy anaerobic chamber with a gas mixture of 10% hydrogen, 5% carbon dioxide, and 85% nitrogen before the colonies were counted. The transformation efficiency was calculated by dividing the number of transformants for the tested plasmid by the number of transformants for the pORI28 control plasmid.The average limit of detection of the killing assay, calculated as 1/(no. of transformants for the control plasmid) was 7 × 10−7 for E. coli, 4 × 10−7 for Salmonella, and 2 × 10−3 for S. thermophilus. The high transformation efficiency for Salmonella was achieved by purifying the pCRISPR plasmids, the pCas3 plasmid, and the pCasA-E plasmid individually from SB300A#1.Mixed-culture transformation assay. The transformation assay for mixed cultures resembled that for the pure culture with a few notable differences. Cultures of E. coli K-12 and E. coli B strains harboring pCas3 and pCasA-E (or pCasA-E′) were grown separately to an A600 of ~0.8, and then equal numbers of cells were mixed from the back dilutions prior to preparing the culture for electroporation. An aliquot of the resuspended cell mixture (50 µl) was then transformed with the pCRISPR plasmid, pCRISPR encoding the indicated spacer, or a defined mixture of both plasmids for a total of 100 ng. The transformed cells were recovered in 300 µl of SOC medium for 90 min. After the recovery period, 200 µl of different dilutions of the cells were plated on LB agar with inducers and appropriate antibiotics. The ratio of blue (E. coli B) to white (E. coli K-12) colonies on the sample plate was divided by the same ratio on the pCRISPR plate, yielding the normalized ratio. To normalize for experimental variability in transformation efficiency, the same batches of cell mixtures prepared for electroporation were transformed with each tested plasmid mixture and the pCRISPR control plasmid.Analysis of escape mutants. Colonies from the transformation assay with the α-ftsA plasmid (pCB304) were inoculated into 5 ml of LB medium with appropriate antibiotics and inducers. Growth was assessed based on the A600 after 13.5 h of growth. Cultures exhibiting measurable growth (A600 > 0.01) were stored as glycerol stocks. Plasmids were then isolated from each escape mutant, and equal amounts of DNA were resolved by agarose gel electrophoresis. Each isolated set of plasmids was also transformed into E. coli K-12 and plated on LB agar containing one of the three antibiotics. Finally, the plasmid mixture from each escape mutant was sequenced using primers that specifically bind within the PBAD promoter or the double terminator of the α-ftsA plasmid. To analyze the protospacers, approximately 400 bp surrounding the protospacer within the ftsA gene of the escape mutant was PCR amplified and subjected to sequencing.Statistical analyses. All P values were calculated using the Student t test, assuming log-normal distributions, two tails, and unequal variances.

Article TitleProgrammable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system inEscherichia colias a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms.


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