MATERIALS AND METHODSBacterial strains, plasmids and mediaAll bacterial strains and plasmids used in this study are listed in Supplementary Table S1. Lactobacilli and their derivatives were cultured at 37ºC under hypoxic conditions (5% CO2, 2% O2) in deMan Rogosa Sharpe (MRS) medium (Difco; BD BioSciences). Lactococcus lactis NZ9000 was used as a general cloning host, cultured statically at 30ºC in M17-broth (Difco; BD BioSciences) supplemented with 0.5% (w/v) glucose. Electrocompetent cells of the LAB used in this study were prepared as described before (36–38). When needed, antibiotics were supplemented at the following concentrations: 5 μg/ml erythromycin and chloramphenicol for L. reuteri ATCC PTA 6475 and L. lactis strains, and 25 and 10 μg/ml tetracycline for L. reuteri and L. lactis, respectively.Reagents and enzymesAll modification enzymes were purchased from Fermentas. Enzyme mixes for Gibson assembly were prepared in a manner identical to that previously described (39). Polymerase chain reaction (PCR) amplifications for cloning purposes were performed with Phusion Hot Start II Polymerase (Fermentas), and PCR amplifications for screening purposes were performed with Taq DNA Polymerase (Denville Scientific). Pellet Paint Co-Precipitant (Novagen) was used to concentrate DNA for Gibson assembly or conventional T4 DNA ligase cloning. Oligonucleotides were purchased from Integrated DNA Technologies.Construction of vectors for CRISPR–Cas9 selection in L. reuteri 6475All oligonucleotides can be found in Supplementary Table S2. pCAS9 and pCRISPR, both kindly provided by Luciano Maraffinni (The Rockefeller University, New York, USA), were used as template DNA to construct derivatives for use in L. reuteri. The backbone of pNZ9530 was amplified with oVPL112-oVPL113 whereby the genes encoding NisR and NisK were excluded yielding a 4.8-kb fragment. The sequence containing the tracrRNA, cas9 and the direct repeats was amplified from pCAS9 with oVPL114-oVPL115 (5-kb amplicon), whereby the resulting amplicon has on the proximal ends 40 bases complementary to the pNZ9530 amplicon generated by oVPL112-oVPL113. Amplicons were precipitated by Pellet Paint Precipitation, and quantified by Qubit Fluorometric Quantitation (Life Technologies). Both amplicons were fused by Gibson assembly, followed by transformation in L. lactis NZ9000. Fusion of both amplicons was confirmed by restriction digest analysis of plasmid DNA, followed by Sanger sequencing to confirm the integrity of the DNA sequence. The resultant construct was named pVPL3004.For cloning the direct CRISPR repeats, we first constructed a derivative of pNZ8048 in which we replaced the gene coding chloramphenicol resistance (cm) with a gene coding tetracycline resistance (tet). This would allow us to use this vector in combination with vectors that encode erythromycin and chloramphenicol resistance. We amplified the backbone of pNZ8048 with oVPL362-oVPL363, located upstream and downstream, respectively, of the cm gene in pNZ8048. The tet gene was amplified with oVPL360-oVPL361 using pORI19Tet (kind gift from Robert Britton, Michigan State University) as template. Both amplicons were precipitated and quantified as described above, and mixed at a 1:1 molar ratio (vector:insert) followed by ligation and electroporation in L. lactis NZ9000. Plasmid DNA derived from tetracycline-resistant colonies was digested with NcoI, an enzyme that digests in the pNZ8048 multiple cloning site and internal to the tetracycline resistance gene, to confirm insertion. The resultant plasmid was named pVPL3112. We constructed a derivative of the RecT expression plasmid pJP042 in which we replaced the gene coding for erythromycin with a gene coding for chloramphenicol resistance to yield pVPL3017. The backbone of pJP042 and the cm gene were amplified with oVPL154-oVPL155 and oVPL156-oVPL157, respectively, and fused by Gibson assembly. The recT gene (locus tag HMPREF0536_0521) is derived from a L. reuteri 6475 prophage (15).Next, the backbone of pVPL3112 was amplified with oVPL309-oVPL310, and the direct CRISPR repeats were amplified with oVPL320-oVPL321. Both amplicons were precipitated and quantified as described above, followed by blunt-end ligation (1:1 molar ratio vector:insert). We confirmed by sequence analysis insertion of the CRISPR repeats yielding pVPL3115, which for clarity is also referred to as pCRISPRctrl.A derivative of pCRISPRctrl was prepared to target the lacL locus in L. reuteri 6475 (see Supplementary Table S2 for oligonucleotides used). Briefly, pCRISPRctrl was digested with BsaI, which cleaves internal to the two CRISPR repeats, followed by gel purification (Fermentas gel purification kit). This allows cloning of a fragment with clamps complementary to the BsaI site to yield a plasmid that contains CRISPR-target sequence-CRISPR. To this end, a pair of complementary oligonucleotides (oVPL151-oVPL152), identical to the 30-bp lacL target region, were annealed generating a double-stranded fragment with overhangs complementary to pCRISPRctrl digested with BsaI. DNA was mixed at a 1:1 molar ratio followed by overnight ligation, pellet paint precipitation and transformation in L. lactis NZ9000. We confirmed by sequence analysis insertion of the lacL protospacer, yielding pCRISPRlacL. In an analogous manner, we constructed pCRISPRsrtA and pCRISPRsdp6 by cloning the complementary oligonucleotides oVPL447-oVPL448 and oVPL453-oVPL454, respectively. The integrity of all pCRISPR plasmids constructed in this study was confirmed by sequence analysis.CRISPR-assisted oligonucleotide genome editing in L. reuteriWe established a single-step and a more robust dual-step approach for CRISPR-assisted oligonucleotide genome editing in L. reuteri, whereby we used the lacL gene (locus tag HMPREF0536_0317), the srtA gene (locus tag HMPREF0536_0973) and the sdp6 gene (locus tag HMPREF0536_0710) as targets to establish proof-of-concept. Cells harboring both pVPL3004 (expressing Cas9 and tracrRNA) and pVPL3017 (RecT expression plasmid) were made competent as previously described (15), with the exception that the growth media contained 5 μg/ml erythromycin and 5 μg/ml chloramphenicol. For the one-step procedure, cells were co-transformed with 100 μg recombineering oligonucleotide (oVPL153_lacL or oVPL449_srtA or oVPL455_sdp6) and 100 ng of the corresponding pCRISPRtarget (pCRIPSRlacL or pCRISPRsrtA or pCRISPRsdp6, respectively). Each of the recombineering oligonucleotides was also combined with pCRISPRctrl, which produces a crRNA that does not have homology to the L. reuteri chromosome. Each of the recombineering oligonucleotides changes upon incorporation five adjacent bases: oVPL153_lacL (CGGGG to TAATA), oVPL449_srtA (AAGGT to TGACA), oVPL455_sdp6 (GGCAG to CTAGC). The protospacer-adjacent site NGG is thus disrupted in all targets, and incorporation of each recombineering oligonucleotide yields in-frame stop codons (see Supplementary Table S1). The pCRISPRtarget plasmids will guide cleavage by Cas9 of the chromosomal target regions if these are not edited by each of the ssDNA recombineering oligonucleotides. After electroporation and recovery, cells were plated on MRS plates with double antibiotic selection to select for pVPL3004 (5 μg/ml erythromycin) and the pCRISPR plasmid (25 μg/ml tetracycline). Colonies were screened by mismatch amplification mutation assay-PCR (MAMA-PCR) (40). MAMA-PCR for the targets lacL, srtA and sdp6 was performed with oligonucleotides oVPL347-oVPL348-oVPL349, oVPL468-oVPL469-oVPL470 and oVPL474-oVPL475-oVPL476, respectively.For the dual-step procedure, cells were transformed with 100 μg oVPL153_lacL followed by overnight recovery in 40 ml MRS harboring 5 μg/ml erythromycin. This overnight recovery step provides additional replication cycles during which oVPL153_lacL can be incorporated in the chromosome, thereby generating a larger population of mutant genotypes. The next day cells were subcultured in MRS to OD600 = 0.1 harboring 5 μg/ml erythromycin, and competent cells were prepared as described previously, but RecT was not induced prior to transformation. Competent cells were transformed with 100 ng pCRISPRlacL or pCRISPRctrl, followed by 2-h recovery in MRS. Plating and screening were performed as described for the one-step procedure. Studies to generate deletions were performed with the dual-step procedure. For both the single-step and the dual-step approach, data were expressed as the level of tetracycline-resistant colony forming units (tetR cfu) per 108 viable cells. It needs to be noted that actual viability levels after electroporation and recovery ranged between 4×108 and 1×109 total cells.CRISPR-assisted targeted codon saturation mutagenesisFor codon saturation mutagenesis, a dual-step approach was performed as described above. The recombineering step was performed with 100 μg oVPL627_NNK. Incorporation of this oligonucleotide yields a single base change (silent mutation) in the PAM region of srtA. Four adjacent mismatches (all silent mutations) are predicted to evade the mismatch repair system, and adjacent to these mismatches are bases that make up a degenerate codon (NNK; N = A/T/G/C, K = T/G). The NNK triplet can yield 32 different codons encompassing all 20 amino acids. In the second step, cells were transformed with 100 ng pCRISPRsrtA. A total of 180 tetracycline-resistant colonies were screened by MAMA-PCR with oligonucleotides oVPL628-oVPL629-oVPL630. Wild-type genotypes yield a 600-bp fragment and incorporation of oVPL627_NNK yielded a 300-bp fragment. Colonies yielding an amplicon of 300 bp were subjected to a second PCR with oligonucleotides oVPL468-oVPL469, and submitted for sequence analysis.
Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as theStreptococcus pyogenesCRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacteriumLactobacillus reuteriATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in theL. reuterichromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.