Materials and Methods

A functional type II-A CRISPR–Cas system fromListeriaenables efficient genome editing of large non-integrating bacteriophage

MATERIALS AND METHODSBacterial strains, bacteriophages, plasmids, and primer L. monocytogenes and L. ivanovii strains were cultivated in 1/2 BHI medium at 30°C. E. coli XL1-blue and S. aureus ATCC19685 were cultivated in LB medium at 37°C. Phages B025, B035, B056, P40, and A511 were propagated on L. ivanovii WSLC 3009 at 30°C, B054 was propagated on WSLC 3009 at 19°C, and P35 was propagated on L. monocytogenes Mack at 20°C. Phage infection assays were carried out using the soft agar overlay method. Briefly, 10 μl phage dilution was mixed with 200 μl stationary host culture in 4 ml LC soft agar (10 g/l tryptone, 5 g/l yeast extract, 10 g/l glucose, 7.5 g/l NaCl, 10 mM CaCl2, 10 mM MgSO4, 0.4% agar) at 46°C and poured onto agar plates (1/2 BHI plates for B025, B035, B054, B056, P40 and A511; LC plates for P35). Plaque-forming units (pfus) were quantified at six (B054) or one (all other phages) days post infection. All plasmids and primers can be found in Supplementary Tables S1 and S2.Phage adsorption assaysTo quantify phage adsorption, 490 μl of SM-Buffer containing 0.02% Tween20 and 2 mM CaCl2 were mixed with 109Listeria cells. 10 μl of phage dilution containing 107 phages and 500 μl 1/2 BHI medium were added. Tubes without Listeria cells served as phage input control. Samples were incubated for 10 min on an overhead rotator at 20°C and centrifuged for 2 min (12 000 × g, 4°C). Supernatants were transferred to new reaction tubes and pellets suspended in 1 ml SM buffer on ice. Serial dilutions of both fractions were prepared and pfus quantified.Cell wall decoration with fluorescent affinity proteinsCell wall-binding domains (CBDs) derived from the endolysins of Listeria phage A500 and S. aureus phage 2638A have previously been engineered as GFP (GFP-CBD500) or dsRed (dsRed-CBD2638A) fusion proteins that allow for selective staining of their respective host (see (28)): 0.5 ml bacterial culture (OD = 1) was harvested (1 min, 7500 g, 4°C), resuspended in 100 μl SM-buffer containing 10 μg of dsRed-CBD or GFP-CBD protein, incubated for 2 min at RT on an overhead rotator, washed twice with 1 ml cold SM-buffer, and finally suspended in 50 μl SM-buffer. 4 μl of stained cultures were spotted onto a microscopy slide and visualized on a Leica TCS SPE confocal system (Leica Microsystems, Germany) equipped with a HCX PL FLUOTAR 100.0 × 1.30 oil objective.BioinformaticsHomology searches were performed using BLAST (29), weblogo was used for visualization of the PAM (30), and cas gene homologies were identified using HHPred (31). To identify putative tracrRNA sequences, CRISPR repeats were aligned with the complete cas-gene region using BLASTn. Identified regions of homology were analyzed for the presence of a bacterial promoter (using BPROM, a sigma70 promoter prediction software) and terminator (using the ARNold software, in the 5′ and 3′ regions, respectively (32–35). Multiple alignments of Cas9 proteins were performed using CLC Workbench (version 9.5.4; Qiagen; settings: progressive alignment, gap open cost = 10, gap extension cost = 1, end gap cost = as any other). Information on the proteins used to create the alignment can be found in Supplementary Table S4. Phylogenetic trees were constructed using CLC Workbench (tree construction method = neighbor joining, protein distance measure = Jukes-Cantor, bootstrap analysis = 100 replicates).Transformation of Listeria Listeria electrocompetent cells were prepared according to a modified protocol by Monk et al. (36). 40 ml 1/2 BHI + 0.5 M sucrose were inoculated with 2 ml stationary phase culture and incubated at 30°C (shaker, 180 rpm) until an OD600nm of 0.2 was reached. Each liquid culture was distributed into four 10 ml aliquots and cells were grown for 2 h at 30°C in the presence of different concentrations of penicillin G (5, 10, 25, 50 μg/ml). For transformation of WSLC 3009, WSLC 1042, and Mack, 10 μg/ml lysozyme was added during the last 10 minutes of incubation. Cells were chilled on ice for 5 min, transferred to a 50 ml Falcon tube, and pelleted by centrifugation (4000 × g, 10 min, 4°C). The pellet was washed once with 1.5 ml and once with 1 ml of cold sucrose-glycerol washing buffer (SGWB; 0.5 M sucrose, 10% glycerol, pH 7.4) before final resuspension (60 μl SGWB per transformation). 1.5 μg plasmid DNA were mixed with 60 μl of electrocompetent cells and transferred to a cold 2 mm electroporation cuvette. Listeria cells were electroporated (2.2 kV/cm 400 Ohm, 25 μF) using a Gene Pulser™ (BioRad Laboratories) and recovered with 1 ml warm 1/2 BHI + 0.5 M sucrose at 30°C for 90 min. Cultures were plated on 1/2 BHI agar plates supplemented with antibiotics (50 μg/ml kanamycin, 5 μg/ml erythromycin, or 10 μg/ml chloramphenicol).Gene deletion in ListeriaGeneration of cas gene deletion mutants (ΔCas(LivCR-1), ΔCas(LivCR-2) and ΔΔCas(LivCR-1/2)) was performed using splicing-overlap-extension (SOE) PCR (37,38), followed by allelic exchange mutagenesis. SOE primers were designed to amplify ∼500 bp sequences flanking the cas gene regions of interest. Flanking fragments were fused using SOE-PCR with primer P76/P79 for ΔCas(LivCR-1) and P80/P83 for ΔCas(LivCR-2). Editing templates were digested, ligated into the pAULA, and transformed into E. coli XL1 blue (39). The editing template to construct the WSLC 30167 ΔBREX mutant was synthesized (Thermofisher Scientific), amplified with primers P100 and P101, and cloned into pAULA. Sequenced editing vectors were transformed into WSLC 30167 by electroporation. Homologous recombination of the plasmid with the genome was selected for by shifting to non-permissive conditions (39°C). After six passages, the antibiotic resistant strains were inoculated into 1/2 BHI without antibiotics at permissive temperatures (30°C). After another five passages, single colonies were screened for loss of erythromycin resistance. Sensitive colonies were screened by PCR for deletion of target genes and PCR products sequenced.Construction of LivCRISPR-1 pre-crRNA expressing vector pLRSR scrA DNA fragment containing the endogenous leader sequence of LivCRISPR-1 (220 bp sequence upstream of the first repeat unit of LivCRISPR-1) followed by one repeat-spacer-repeat (RSR) unit and 350 bp of sequence downstream of the last endogenous repeat unit was synthesized. This synthetic CRISPR array was cloned into the E. coli-Listeria shuttle vector pLEB579 (40) using restriction enzyme XbaI to yield the pre-crRNA vector pLRSR scr. The spacer region of pLRSR scr contains two BsaI sites that enable incorporation of any spacer sequence of choice using annealed oligonucleotides, essentially as described by Jiang et al. (41). Appropriate BsaI overhangs need to be reconstituted to generate functional RSR units upon ligation with BsaI-digested pLRSR scr (see Figure ​Figure3A). For3A). For annealing, 50 μl oligonucleotide pairs (4 μM each) were mixed in T4 ligase buffer, heated to 100°C for 10 min, and slowly cooled to RT in a heating block. Annealed spacers were ligated with BsaI-digested pLRSR scr, transformed into E. coli XL-1 blue cells, and grown on LB plates supplemented with 300 μg/ml erythromycin. All pLRSR-derived plasmids and oligonucleotide sequences can be found in Supplementary Tables S1 and S2.Open in a separate windowFigure 3.Programmable LivCRISPR-1 Cas9 targets bacterial DNA and is transferrable to L. monocytogenes. (A) Schematic representation of the pLRSR scr plasmid used as a backbone to express LivCRISPR-1 pre-crRNAs. Spacer sequences are inserted into BsaI-digested pLRSR scr using annealed oligonucleotides with appropriate 3′-overhangs. (B–D) Self-targeting assays: To quantify CRISPR activity, transformation efficiencies of DNA polymerase targeting pre-crRNA plasmids were compared with non-targeting controls (pLRSR scr, CTRL). Interference activity of LivCRISPR-1 was assayed in (B) WSLC 30167 wild type, LivCRISPR-1/2 deletion mutants, and cas9/tracrRNA reconstituted strains. Furthermore, LivCRISPR-1 cas9 and tracrRNA were transferred to (C) L. ivanovii ssp. ivanovii (WSLC 3009) and (D) L. monocytogenes (WSLC 1042) strains and tested for activity. Data are presented as mean ± SD from three biologically independent experiments.Transfer of cas9 and tracrRNA into ListeriaFor genomic expression of LivCRISPR-1 cas9 and tracrRNA from their endogenous promoter, the cas9 5′-region (360 bp), cas9 gene, and tracrRNA were amplified from using primers P1 and P2 and cloned into the integrative plasmid pIMK (42) to yield pIMK Pend cas9. For overexpression, cas9 and tracrRNA were amplified without the cas9 5′ region using primers P3 and P4 and cloned into pIMK2 to yield pIMK2 Phelpcas9. Listeria strains were transformed with both plasmids and integration selected for with 50 μg/ml kanamycin.CRISPR-Cas-mediated targeting of bacterial and phage genomic DNATo target bacterial gDNA, Listeria cells were electroporated with 1.5 μg of pLRSR plasmid expressing a pre-crRNA that targets the DNA polymerase I gene of the transformed Listeria strain (self-targeting vector). As control, 1.5 μg of a non-targeting pre-crRNA plasmid (pLRSR scr) was used. Transformed cells were grown on 1/2 BHI plates supplemented with 5 μg/ml erythromycin for 48 h at 30°C, colony forming units quantified by plating serial dilutions, and relative transformation efficiencies calculated. To target phage genomic DNA, LivCRISPR-1 cas9 and tracrRNA expressing propagation strains were transformed with pre-crRNA plasmids targeting late genes of phages P40 (pLRSR P40; targets the putative tail tape measure protein gp14), A511 (pLRSR A511; targets the putative tail tip protein gp028), and P35 (pLRSR P35; targets the putative tail tape measure protein gp14). Plasmids were constructed as described in Supplementary Tables S1 and S2. Artificial bacteriophage-insensitive mutants (BIMs) were tested for phage sensitivity using spot-on-the-lawn assays: 200 μl over-night culture was mixed with 4 ml soft-agar and poured on bottom-agar in a 10 cm petri dish. Once solidified, serial phage dilutions (10 μl) were spotted on this plate and incubated over-night.LivCRISPR-1-assisted site-directed mutagenesis of phage A511Eight editing plasmids with homology regions flanking both sides of the protospacer and PAM sequence were constructed using shuttle vector pSK1 as a backbone. Four plasmids contained one mutation in the PAM (NNACAC to NNATAC) and 400, 250, 150 or 50 bp homology arms on each side. The remaining four plasmids contained four additional silent mutations (see Figure ​Figure5B)5B) and the same homology arms. Flanking regions were amplified by PCR using primers containing the mutated spacer/PAM sequences, purified, and assembled with the pSK1 plasmid backbone using the Gibson Assembly method (NEBuilder HiFi DNA Assembly Cloning Kit) to yield the editing plasmids. A511 gp97 crRNA targeting construct was cloned by incorporation of annealed oligonucleotides P13 and P14 into BsaI-digested pLRSR scr to yield pLRSR A511. Mutant phages were isolated using a one-step protocol: The WSLC 3009::Phelp cas9 strain containing pSK1-derived editing plasmids and the A511-targeting pre-crRNA plasmid (pLRSR gp97) was infected with serial dilutions of A511 wild type phages using the soft-agar overlay assay. Two phage plaques were sequenced to validate genotype.Open in a separate windowFigure 5.LivCRISPR-1-assisted site-directed mutagenesis of virulent phage genomes. (A) Schematic representation of the CRISPR-assisted phage genome engineering strategy. Left: The host strain carries an editing plasmid that mediates double homologous recombination with the incoming / replicating phage genome, producing a mixture of wild type and recombinant phages. Right: Engineered phages are enriched by selective, LivCRISPR-1-mediated cleavage of wild type genomes and concomitant replication of the recombinant phage. pEdit = editing plasmid. (B) Strategy for site-directed mutagenesis in gp97 of phage A511. Protospacer and PAM sequences in the targeted region of A511 gp97 and the corresponding editing plasmids for the incorporation of one or five silent point mutations are shown. Mutated base pairs are indicated red. Homology regions shared between the editing plasmid and the A511 gp97 gene are indicated (dashed crosses). (C) L. ivanovii WSLC 3009::Phelp cas9 pLRSR gp97 strains were transformed with the indicated editing plasmids, infected with A511 wild type phages, and the efficiency of plating (eop) was determined. As a control, WSLC 3009 wild type strains were transformed with the same editing plasmids and eop determined. The size of the homology arms varied between 0 bp (pSK1 empty control) and 400 bp and the corresponding editing plasmids encoded either one or five silent point mutations (1mut and 5mut, respectively). Data are presented as mean ± SD from three biologically independent experiments.Construction of lysostaphin-encoding A511::lst phagesPre-crRNA expression plasmids targeting the endolysin or major capsid gene of A511 were constructed as described above using primers P62 and P63 (pLRSR A511 ply) or P64 and P65 (pLRSR A511 cps), respectively. The editing plasmids containing flanking homology arms and the lysostaphin-hexahistidine (his6) gene sequence (771 bp + ribosomal binding site: GAGGAGGTAAATATAT) were assembled into the pSK1 backbone as described in Supplementary Tables S1 and S2. Silent mutations were subsequently introduced into the PAM motif of these editing plasmids using site-directed mutagenesis to allow for CRISPR escape of recombinant phage genomes. The final editing plasmids pSK1 ply511 lst-his6 and pSK1 cps511 lst-his6 mediate integration of the lysostaphin-his6 gene downstream of the A511 endolysin (ply511) and major capsid gene (cps511), respectively. First, L. ivanovii WLSC 3009 was transformed with either of the two editing plasmids and infected with A511 wild type phage using the soft-agar overlay technique to obtain semi-confluent lysis. The obtained phage lysate was subsequently titered on WSLC 3009::Phelp_cas9 pLRSR_A511ply or WSLC 3009::Phelp_cas9 pLRSR_A511 cps. Four candidate plaques were picked for each phage and assayed for the correct genotype using PCR and sequencing (see Supplementary Figure S6).Infection of Listeria and Staphylococcus co-culturesStationary phase cultures of WSLC 3009 and/or ATCC19685 were diluted in 1/2 BHI to obtain an OD of 0.1 (OD = 0.05 of each strain for co-culture infections). 15 ml of the cultures were incubated at 30°C for 1 h before phage addition (t = 0) at a multiplicity of infection (moi) of 0.03. Infected cultures were incubated (30°C) and optical density monitored for 320 minutes. In addition, serial dilutions were plated on selective Oxford (Listeria) and Baird-Parker (Staphylococcus) agar plates (biolife) at 0, 100, 200 and 300 min post infection. Cfus were counted after 24 h incubation at 30 or 37°C, respectively.Purification of lysostaphin-his6 from phage infected culturesOne liter of prewarmed 1/2 BHI medium was inoculated with 20 ml of a WSLC 3009 overnight culture. Phages (A511::lst1 or A511::lst2) were added to a final concentration of 1 × 105 pfu/ml. After 3–4 h at 30°C more phages were added to a final concentration of 1 × 107 pfu/ml and incubated for another 3 h. Cleared lysates were centrifuged (10 000 × g, 10 min) and supernatants incubated with 10 ml Ni-NTA Superflow resin (Qiagen) at 4°C for 30 min on an overhead rotator. Resin was transferred to MicroBiospin columns (Bio-Rad) and washed extensively with buffer A (500 mM NaCl, 50 mM Na2HPO4, 5 mM imidazole, 0.1% Tween 20 pH 8.0). His-tagged lysostaphin was eluted using Buffer B (500 mM NaCl, 50 mM Na2HPO4, 250 mM imidazole, 0.1% Tween 20 pH 8.0). Pooled fractions were dialyzed twice against dialysis buffer (NaH2PO4, 120 mM NaCl pH 8.0, 0.01% Tween 20) and samples assayed by SDS-PAGE.

Article TitleA functional type II-A CRISPR–Cas system fromListeriaenables efficient genome editing of large non-integrating bacteriophage


CRISPR–Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR–Cas system from the genusListeriaand demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome ofL. ivanoviisubspecieslondoniensisthat uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1cas9and trans-activating crRNA intoListeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integratingListeriaphages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-rangeListeriaphage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targetsStaphylococcuspeptidoglycan. In bacterial co-culture, the armed phages not only killedListeriahosts but also lysedStaphylococcuscells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR–Cas-assisted phage engineering, beyond single pathogen control.

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