Genome Engineering of Virulent Lactococcal Phages Using CRISPR

Research Article pubs.acs.org/synthbio

Genome Engineering of Virulent Lactococcal Phages Using CRISPRCas9 Marie-Laurence Lemay,† Denise M. Tremblay,† and Sylvain Moineau*,† †

Département de biochimie, de microbiologie, et de bioinformatique, Faculté des sciences et de génie, Félix d’Hérelle Reference Center for Bacterial Viruses, and Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, Québec G1V 0A6, Canada S Supporting Information *

ABSTRACT: Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described. KEYWORDS: virulent bacteriophages, lactic acid bacteria, genome engineering, CRISPR-Cas9, homologous recombination, double-strand DNA break repair

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Cas) have proven useful for genetically engineering a plethora of organisms (reviewed in ref 13), including phages.9−11 CRISPR-Cas systems are natural defense mechanisms found in many prokaryotes that provide sequence-specific protection against invasion by foreign nucleic acids.14,15 This immune response depends on complementary base pairing between the spacer in the CRISPR array (the effective form being a crRNA) and a matching protospacer in the genome of the invader.14,16−18 CRISPR-Cas systems are extremely diverse in nature and are classified according to their associated Cas proteins.19 Class 1 systems require the activity of a multiprotein complex, whereas class 2 systems have a single protein effector. The on-target cleavage provided by both classes has been repurposed for genome engineering. As such, class 1 subtype IE CRISPR-Cas systems from E. coli and Vibrio cholerae have been used to modify lytic phage genomes.9,11 Similarly, the endogenous class 2 subtype II-A system from Streptococcus thermophilus was used to modify the genome of a virulent phage.10 The Cas9 protein is the signature nuclease of the type II systems (class 2).19,20 Type II systems have in common the

n the era of metagenomics, the availability of viral sequence data is continuously increasing. Currently, over 2.79 million genes code for viral proteins, but 75% of these have no known function and no homologue in public databases.1−5 The increasing gap between the number of viral genes and the assignment of gene function is mainly due to the abundance of viruses,6 their diversity,7 and difficulties in studying them. Annotations of structural proteins greatly outnumber those of nonstructural proteins because they are more conserved and easier to study with techniques such as SDS-PAGE, mass spectrometry, and electron microscopy.8 Viruses are obligate parasites as they depend on the host cellular machinery for their multiplication. A viral genome can be modified and studied in vivo only while inside a host, complicating functional studies. Genome manipulation of virulent bacterial viruses (bacteriophages or phages) is also a challenge since their genome never integrates into the bacterial chromosome. Moreover, the tight time frame to engineer virulent phages is dictated by the short duration of the infection cycle. The lack of tools to efficiently edit the genome of virulent phages partly explains why many nonstructural proteins are still uncharacterized.9−12 Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR© 2017 American Chemical Society

Received: December 22, 2016 Published: March 21, 2017 1351

DOI: 10.1021/acssynbio.6b00388 ACS Synth. Biol. 2017, 6, 1351−1358

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ACS Synthetic Biology

Figure 1. New spacer cloning in pL2Cas9. (A) Schematic representation of phage p2 genomic organization. The gene expression modules are designated by colored boxes. Late-expressed phage genes during the infection of L. lactis are enclosed in a red box, while early- and middle-expressed genes are enclosed in green and yellow boxes, respectively. Black arrows indicate genes of unknown function and white arrows indicate genes with assigned function. (B) Orf47 is a putative protein of no known function. (C) The nucleotide sequence of orf47 in the phage p2 genome is partially shown. The targeted PAM and protospacer are in black and light gray, respectively. To generate a CRISPR-Cas9-mediated double-stranded break in orf47, the 30 bp corresponding to the protospacer were cloned as a spacer between two repeats (diamonds) in the crRNA of pL2Cas9.

species. Yet, it is still not possible to readily manipulate the phages of this host. The use of CRISPR-Cas9 for genome engineering has only been described in two lactic acid bacteria (LAB): the probiotic Lactobacillus reuteri31 and the starter culture S. thermophilus.10 CRISPR-mediated immunity to phages was first demonstrated in S. thermophilus.14,15,32 Aside from a plasmid-encoded type III system, no CRISPR-Cas system was identified in L. lactis.33 In this study, CRISPR-Cas9 technology is introduced into L. lactis. The S. pyogenes type II-A CRISPR-Cas system is demonstrated to function in L. lactis, efficiently editing the previously intractable genome of virulent phage p2. More precisely, targeted gene perturbation, single base modification, and insertion into orf47, which encodes a conserved nonstructural protein, was achieved. Although orf47 is part of the sk1 group core-genome, it has no known function.34 The reproducibility of this method was demonstrated by targeting and disturbing additional p2 genes: orf 24, orf42, and orf49. The CRISPR-Cas9 tool presented here has the potential to significantly expand our knowledge of phage−host interactions by providing new information about their many uncharacterized proteins.

need of a protospacer adjacent motif (PAM) next to the target site.15,21,22 The PAM is specific to every Cas9 orthologue. Streptococcus pyogenes Cas9 (SpCas9), the most intensely studied Cas protein, requires a 5′-NRG-3′ (where R = G or A) PAM to target a sequence.23,24 SpCas9 can be programmed with a crRNA containing a desired spacer to dictate target specificity.25 It can target virtually any DNA sequence, and the only requirements are PAM recognition and a designed crRNA. SpCas9 has been used in numerous organisms (reviewed in ref 13), but it has never been exploited for phage engineering. Lactococcal phages are natural inhabitants of milk, and because they are mostly unaffected by pasteurization, they pose a risk to milk fermentation processes through infection of industrial strains of L. lactis. Virulent lactococcal phages belonging to the sk1 group are by far the most prevalent in the dairy industry.26,27 Phage p2 is the model for this group and it infects the plasmid-cured strain L. lactis MG1363, itself a model Gram-positive bacterial strain for basic research. Phage p2 belongs to the Siphoviridae family of the Caudovirales order.28 Its 69 nm diameter capsid contains a dsDNA genome of 27 595 bp and 49 predicted open reading frames (orfs). While significant research has been done on the structure of phage p2,29,30 it is still far from being well understoodalmost half of its genes encode proteins with no assigned function. As a model bacterium, L. lactis is amenable to genetic manipulation, and efficient means are available for introducing foreign DNA into most strains. Many expression systems have also been developed to produce recombinant proteins in this

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RESULTS AND DISCUSSION

Introduction of CRISPR-Cas9 into L. lactis MG1363. Plasmid pCas9 carries the gene encoding the Cas9 endonuclease as well as the genetic elements needed to generate the tracrRNA and a crRNA. These genetic elements

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Figure 2. Construction of three homologous recombination templates. (A) To construct pKO47, a 547 bp fragment (left, containing the 3′-end of orf45, orf46, and the 5′-end of orf47) and a 352 bp fragment (right, containing the 3′-end of orf47 and the 5′-end of orf48) were amplified from the phage p2 genome. The external primers had overlaps (green and blue) appropriate for inserting the amplicons into linearized vector using Gibson assembly. The inner primers had complementary overhangs (pink) for annealing. Assembly of the PCR products disrupted the sequence of orf47 (hatched gene). The protospacer (gray) and the PAM (black) are absent from pKO47, preventing CRISPR-Cas9 interference with the generated recombinant phages. The same strategy was used to construct pKO24, pKO42, and pKO49. (B) To construct p47G86C, a 609 bp fragment (left, containing the 3′-end orf45, orf46, and the 5′-end of orf47) and a 366 bp fragment (right, containing the 3′-end of orf47 and the 5′-end of orf48) were amplified from the phage p2 genome using the same external primers. The inner primers were designed to generate a point mutation (∗) removing the PAM from the sequence, thereby preventing CRISPR-Cas9 interference. (C) To construct pHis47G86C, PCR reactions were performed using p47G86C as the template, rather than the phage genome, since p47G86C already had the point mutation to eliminate the PAM. The inner primers had 18 bp overhangs (yellow) to allow fusion of an N-terminal polyhistitine-tag immediately after the start codon.

Efficiency of pNZCas9 and pL2Cas9 Protection against Phage Infection. Because pNZCas9 was successfully transformed into E. coli NEB5α, its efficiency in protecting E. coli against lytic phage infection could be tested. A spacer targeting the gene coding for the phage T4 DNA replication protein repEA was cloned into the crRNA of pNZCas9 to generate pNZCas9-T4, which was introduced into E. coli MG1655. Colonies were selected randomly, and the construction was confirmed by sequencing. The level of phage resistance (interference) provided by pNZCas9-T4 was measured by efficiency of plaquing (EOP). The EOP was calculated by dividing the phage titer obtained on the resistant strain (E. coli MG1655 pNZCas9-T4) by the phage titer obtained on the sensitive strain (E. coli MG1655 pNZCas9). Following infection of the two bacterial strains with virulent phage T4 (biological triplicates), a modest reduction in EOP of 0.71 ± 0.01 was observed.

were all derived from the type II-A CRISPR-Cas system of S. pyogenes SF370.24 Since pCas9 does not replicate in L. lactis, the variant pNZCas9 was generated by cloning the above CRISPRCas9 system into the broad-host-range, high-copy number plasmid, pNZ123,35 and transforming it into E. coli NEB5α (host for cloning). The integrity of pNZCas9 was confirmed by sequencing. The plasmid was then transformed into L. lactis MG1363. All transformation attempts were unsuccessful, as all colonies tested had lost the CRISPR-Cas9 components. These results suggested that Cas9 may be toxic to L. lactis when expressed from a high-copy vector. Decreasing the expression of Cas9 was likely necessary. Another derivative of pCas9 was constructed using the lowcopy number plasmid, pTRKL2.36 The resulting pL2Cas9 was successfully transformed into E. coli and then into L. lactis MG1363. The plasmid was sequenced in its entirety and no mutations were found. 1353


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Figure 3. Targeted genome editing of virulent phage p2. (1) L. lactis MG1363 containing the plasmids pKO47 and pL2Cas9−47 was infected with virulent phage p2. (2) Shortly after the viral DNA enters the cell, the CRISPR-Cas9 system recognizes the targeted protospacer (gray) and PAM (black) located in orf47 and generates a double-stranded break. (3) The lesion is then repaired with the recombination template pKO47, to permanently inactivate orf47. (4) The resulting edited genome lacks the target sequence, therefore avoiding cleavage by the CRISPR-Cas9 system. (5) The recombinant phage p2Δ47 is released through cell lysis. The same principle applies for all the genome modifications described, the only difference being the recombination template used to repair the lesion.

Similarly, the phage resistance provided by pL2Cas9 was investigated in L. lactis. SpCas9 was programmed to cleave a specific region of phage p2 orf47 by cloning a new spacer into the crRNA of pL2Cas9 to generate pL2Cas9−47 (Figure 1). The ability of pL2Cas9−47 to protect L. lactis MG1363 against infection by virulent phage p2 was then tested. The assays were performed at three temperatures: 30 °C, the optimal growth temperature for L. lactis, 37 °C, the optimal growth temperature for S. pyogenes, and 35 °C, an intermediate temperature. In all cases, the reduction in EOP was modest but sufficient to show that the S. pyogenes CRISPR-Cas9 system can interfere with phage infection in L. lactis MG1363. EOPs of 0.49 ± 0.13, 0.31 ± 0.14, and 0.44 ± 0.03 were observed after incubation at 30, 35, and 37 °C, respectively. Sequence Analysis of Escaping Phages. After infecting the transformant L. lactis MG1363 pL2Cas9−47 with phage p2 at 30 °C, 92 phage plaques were picked, and the phage genomic region targeted by CRISPR-Cas9 was analyzed using PCR (primers CB13.42 and orf48_1−2) and subsequent sequencing. No mutations were detected in either the protospacer or the PAM of these seemingly CRISPR-escaping mutants (CEMs). These results were likely due to the weak phage resistance phenotype conferred by pL2Cas9−47, and the plaques were not from CEMs (phages with a mutated protospacer or PAM) but from wild-type phage p2. Genome Engineering of the Virulent Phage p2. To determine if the interference provided by the S. pyogenes CRISPR-Cas9 system in L. lactis was sufficient to readily modify the genome of the virulent phage p2, templates for homologous recombination were provided to facilitate screening for specific mutations. These templates were designed to contain not only a desired mutation and homologous flanking arms to allow recombination, but also to lack the motif targeted by SpCas9 and to prevent further interference by the system (Figure 2). Gene deletions, point mutation within a gene, and insertion of desired nucleotides were successfully achieved, all within the

genome of the virulent lactococcal phage p2 using pL2Cas9 (Figure 3). Deletion. To create a deletion in orf47 of phage p2, a recombination template was constructed by Gibson assembly (see Methods) and cloned into pNZ123 (in L. lactis) to generate pKO47 (Figure 2A). Of note, the plasmid pNZ123 is compatible with pTRKL2, the vector backbone of pL2Cas9. After confirmation of pKO47 by sequencing, the adapted gene editing tool pL2Cas9−47 was transformed into L. lactis MG1363 containing pKO47. The resulting strain (L. lactis MG1363 pKO47 pL2Cas9−47) was then challenged with increasing dilutions of phage p2 in order to obtain distinct lysis plaques. Three randomly selected phage plaques were analyzed by PCR. Two plaques showed two distinct bands corresponding to a mixed population of wild-type and recombinant phages (data not shown). The third plaque showed a single band corresponding to the expected deletion of 76 bp in orf47 (Figure 4). Still, we assumed that this third plaque also contained a mixed population of phages, but with a higher proportion of recombinant phages, as readily detected by a shorter PCR product. Therefore, the recombinant phage was purified by three sequential rounds of infection on the same strain. This methodology ensured the removal of any remaining wild-type phages. The deletion in orf47 resulted in a truncated protein retaining only eight N-terminal amino acids (out of 43) from Orf47. Full genome sequencing of the phage mutant p2Δ47 detected no additional mutations, indicating no genetic compensation and, more importantly, no off-target activity with this CRISPR-Cas9 adapted tool. Disrupting this gene indicated that it is a bona f ide nonessential gene for phage p2, under the conditions tested. This phage mutant will allow investigation of the role of Orf47 during the phage infection process. Taken altogether, it is possible to recover a recombinant phage despite the modest phage resistance phenotype provided by pL2Cas9−47. Of note, a similar experiment was performed 1354


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of p2His47G86C caused loss of the His-tag, but purification with L. lactis MG1363 pL2Cas9−47 addressed this problem. Both the 18 bp insertion and the point mutation in recombinant phage p2His47G86C were confirmed by sequencing. Targeting Other Phage p2 Genes. To evaluate the robustness of pL2Cas9 for modifying the genome of the virulent phage p2, additional mutations were generated. Using the same strategy as described for pL2Cas9−47 (Figure 1), new spacers were cloned into the crRNA of pL2Cas9 to construct pL2Cas9−24, pL2Cas9−42, and pL2Cas9−49 to target orf 24, orf42, and orf49, respectively. To delete these three genes of unknown function, recombination templates pKO24, pKO42, and pKO49 were constructed using the same method as for pKO47 (Figure 2A). All recombination templates and crRNA were confirmed by sequencing. L. lactis MG1363 pKO24 pL2Cas9−24, L. lactis MG1363 pKO42 pL2Cas9−42, and L. lactis MG1363 pKO49 pL2Cas9− 49 were independently infected with phage p2. One randomly selected phage plaque was selected for each bacterial strain. Mutant phages p2Δ24, p2Δ42, and p2Δ49 were each purified by three rounds of infection on their cognate host. Deletions were analyzed by PCR and subsequent migration on a 2% agarose gel (Figure 4). PCR products were also sequenced to confirm the mutations. No apparent differences in the efficiency of mutation were observed since all phage recombinants were successfully obtained using the same strategy. The 117 bp deletion in virulent phage p2 early expressed gene orf 24 resulted in a truncated protein retaining 26 Nterminal and 13 C-terminal amino acids (out of 78) from Orf24. Full genome sequencing of the phage mutant p2Δ24 showed no other mutation. The 72 bp deletion in the early expressed gene orf42 resulted in a truncated protein retaining 7 N-terminal and 27 C-terminal amino acids (out of 60) from Orf42. The 132 bp deletion in the middle-expressed gene orf49 resulted in a truncated protein retaining only 12 N-terminal amino acids (out of 55) from Orf49. Sequential Mutations. A mutant of phage p2 carrying deletions in multiple genes was generated by sequential recombination coupled with the selective pressure conferred by CRISPR-Cas9. L. lactis MG1363 pKO24 pL2Cas9−24 was first infected with the recombinant phage p2Δ47. The sequence of the resulting recombinant phage p2Δ47Δ24 was confirmed by full genome sequencing. To obtain the mutant p2Δ47Δ24Δ42, L. lactis MG1363 pKO42 pL2Cas9−42 was then infected with phage p2Δ47Δ24. The three deletions in the genome of the resulting phage were confirmed by PCR, agarose gel electrophoresis (Figure 4) and sequencing. L. lactis MG1363 pKO49 pL2Cas9−49 was then infected with p2Δ47Δ24Δ42 to obtain a derivative of phage p2 lacking four genes out of the 49 annotated ones (8%). The four deletions in phage p2Δ8% were also confirmed by PCR, gel electrophoresis (Figure 4) and sequencing. Knockouts of those four genes suggest that they are nonessential under the conditions used.

Figure 4. PCR-based confirmation of deletions in the genome of phage p2 recombinants. PCR products were migrated on a 2% agarose gel, together with 1 kb plus DNA ladder (Life Technologies) (lanes 1, 7, 12, and 18). The deletion in orf 24 was confirmed by the shorter PCR products obtained with mutated phages p2Δ24, p2Δ47Δ24, p2Δ47Δ24Δ42, and p2Δ8% (lanes 3 to 6, respectively), compared to the wild-type phage p2 (lane 2). The deletion in orf42 was confirmed by the shorter PCR products obtained with mutated phages p2Δ42, p2Δ47Δ24Δ42, and p2Δ8% (lanes 9 to 11, respectively), compared to the wild-type phage p2 (lane 8). The deletion in orf47 was confirmed by the shorter PCR products obtained with mutated phages p2Δ47, p2Δ47Δ24, p2Δ47Δ24Δ42, and p2Δ8% (lanes 14 to 17, respectively), compared to the wild-type phage p2 (lane 13). The deletion in orf49 was confirmed by the shorter PCR products obtained with mutated phages p2Δ49 and p2Δ8% (lanes 20 and 21, respectively), compared to the wild-type phage p2 (lane 19).

with phage p2 and L. lactis MG1363 containing only the recombination template pKO47. No recombinant phage was obtained (out of the 45 phage plaques tested), highlighting the requirement for pL2Cas9−47 to efficiently obtain the desired phage mutant. Point Mutation. Using the same strategy as above, the recombination template p47G86C (Figure 2B) was constructed to generate a point mutation within orf47. The point mutation G86C changed the 5′-TGG-3′ PAM to 5′-TGC-3′, preventing SpCas9 from targeting a recombinant phage. L. lactis MG1363 containing both p47G86C and pL2Cas9−47 was infected with phage p2, and a randomly selected phage plaque was purified thrice on the same host to obtain phage p247G86C. The mutation in the recombinant phage genome was confirmed by sequencing the PCR product of orf47. This point mutation showed that a single mutation in the PAM was sufficient for the recombinant p247G86C to bypass the interference activity of CRISPR-Cas9. Insertion. Another recombination template (pHis47G86C) was constructed to introduce an 18 bp sequence corresponding to a polyhistidine-tag at the N-terminus of Orf47, as well as the point mutation G86C (Figure 2C), described above. The mutation changed amino acid 29 from glycine to alanine, which is unlikely to change the activity of Orf47 (data not shown). L. lactis MG1363 containing pHis47G86C and pL2Cas9−47 was then challenged with the virulent phage p2 to engineer the recombinant phage p2His47G86C. After the first round of infection, one randomly selected phage was plaque purified three times using a bacterial strain carrying only pL2Cas9−47. The presence of the recombination template during purification

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CONCLUSION This study shows that the programmable on-target cleavage provided by SpCas9 can be used to reliably generate precise mutations in the genome of the lactococcal virulent phage p2. Although naturally occurring homologous recombination can be co-opted to generate mutations in a phage genome, recombination rates are very low and the selection of a specific 1355


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de Génotypage des Génomes at the CHUL center. For wholegenome and complete plasmid sequencing, libraries were prepared using the Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed on a MiSeq system using a MiSeq reagent kit v2 (Illumina). The sequences were analyzed using Geneious 7.1.4. Construction of pNZCas9. Plasmid pCas924 was a gift from Luciano Marraffini (Addgene plasmid #42876). It was digested with SalI and XbaI, generating two fragments of 3524 bp and 5802 bp, the latter retaining the CRISPR-Cas9 components. Plasmid pNZ123 was amplified with the primers pNZ123_Gibson_F and pNZ123_Gibson_R. Both primers were designed to have overhangs suited for Gibson assembly41 with the appropriate pCas9 fragment. Prior to Gibson assembly, the two fragments to be assembled (5802 bp and PCR product of pNZ123) were purified using the QIAquick PCR purification kit (Qiagen). The assembled product was transformed into E. coli NEB5α, and transformants were isolated on LB Cm 20 plates. Three individual colonies were screened for the presence of pNZCas9. They were grown overnight in fresh media, and their plasmids were isolated. The plasmids were then digested with BsaI, which has two unique restriction sites within the crRNA on both sides of the spacer. Restricted plasmids were separated on a 2% (w/v) agarose gel to confirm that both fragments had been annealed. The CRISPR-Cas9 components were sequenced and aligned with the sequence of pCas9 to verify their integrity. Construction of pL2Cas9. To construct pL2Cas9, plasmid pTRKL2 was amplified with the primers pTRKL2_Gibson_F and pTRKL2_Gibson_R, both located in the lacZ reporter sequence, yielding a 6419 bp fragment. Using the same strategy as described above, pTRKL2 was assembled with the CRISPRCas9 components of pCas9. The assembled product, named pL2Cas9, was transformed into E. coli NEB5α and transformants were isolated on BHI Em 150 plates. Individual colonies were screened for the presence of cas9 by colony PCR using Cas9_S.pyo_F5 and Cas9_S.pyo_R1 primers. Colonies yielding a PCR fragment of the correct size were grown overnight. Plasmids were purified from these cultures and sequenced to confirm the integrity of the CRISPR-Cas9 components. New Spacer Cloning in pNZCas9 and pL2Cas9. Both plasmids were digested with BsaI and purified by precipitation with salts and ethanol. Oligonucleotides PS_T4 and PS_T4_RC were designed to match a protospacer located in the gene repEA of phage T4 and to have ends complementary to the BsaI site to allow cloning into pNZCas9. The oligonucleotides were annealed and ligated overnight into the digested plasmid, using an insert: vector molar ratio of 3:1 with approximately 0.1 μg of total DNA. The resulting pNZCas9-T4 was transformed into E. coli MG1655, host of phage T4. Using the same method, oligonucleotides PS_47 and PS_47_RC were annealed and cloned into pL2Cas9. This spacer is complementary to a protospacer flanked by a PAM and located in orf47 of phage p2 (Figure 1). The resulting pL2Cas9−47 was dialyzed on a membrane and electroporated into L. lactis MG1363, host of phage p2. Likewise, oligonucleotides PS_24 and PS_24_RC, PS_42 and PS_42_RC, as well as PS_49 and PS_49_RC were cloned into the crRNA of pL2Cas9 to generate plasmids pL2Cas9−24, pL2Cas9−42, and pL2Cas9−49, respectively. The presence of the correct spacers was confirmed by sequence analysis of the

mutant can be time-consuming. The method described here is simple to carry out and highly efficient, thereby avoiding largescale plaque screening to obtain the desired recombinant virulent phage. In all cases, we observed equal mutation efficiencies. While not tested here, it is very likely that Cas9 variants could also be used in L. lactis, including the catalytically inactive Cas9, to modulate gene expression.37,38 A total of 4992 PAMs (NRG) are spread across the genome of phage p2 and each orf can be targeted by SpCas9, offering the possibility of studying the functions of these orfs in vivo. Knockout studies are an efficient way to begin elucidating protein function, but they are not suitable for investigating genes essential for phage multiplication. To study the role of essential genes in vivo, nondisruptive mutations must be generated. Being able to fuse a His-tag to a phage protein of interest provides a new tool to purify that protein and identify its binding partners during the phage infection process. Finally, pTRKL2, the vector backbone of pL2Cas9, is stable in many other LAB,36 providing opportunities to edit the genomes of other phages infecting these industrially relevant bacterial strains.

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METHODS Bacterial Strains, Phages, and Growth Conditions. All bacterial strains, phages, and plasmids used in this study are listed in Supplementary Table S1. Phages p2 (GenBank GQ979703) and T4 as well as their hosts were obtained from the Félix d’Hérelle Reference Center for Bacterial Viruses (www.phage.ulaval.ca). The bacterial strain L. lactis MG1363 and its derivatives were grown statically at 30 °C in M17 broth (Oxoid, Ontario, Canada) supplemented with 0.5% glucose (GM17), unless otherwise stated. For solid media, agar (1.0% w/v) was added to GM17 broth. For transformation, electrocompetent cells were prepared as described previously.39 To avoid plasmid loss, chloramphenicol or erythromycin was added to the media to a final concentration of 5 μg/mL (Cm 5 or Em 5). E. coli strains were grown in LB medium or BHI and incubated at 37 °C with agitation. For cloning purposes, chemically competent E. coli NEB5α were purchased (New England Biolabs). When needed, chloramphenicol was added to a final concentration of 20 μg/mL (Cm 20) in LB and erythromycin was added to a final concentration of 150 μg/mL (Em 150) in BHI. For solid media, agar (1.5% w/v) was added to LB or BHI broth. For phage infection, media were supplemented with 10 mM CaCl2 and no antibiotic was added. For double layer plaque assays,40 plates contained a bottom layer of the appropriate medium supplemented with 1.0% (w/v) agar and a top layer of the medium supplemented with 0.75% (w/v) agar. Reagents and Enzymes. Plasmids were purified from overnight bacterial cultures using the QIAprep Spin Miniprep kit (Qiagen). Prior to plasmid extraction, L. lactis cultures were treated with lysozyme (30 mg/mL, 30 min, 37 °C). Restriction enzymes were purchased from Roche Applied Science or New England Biolabs (BsaI). Polymerase chain reactions (PCR) were performed with Taq polymerase (Feldan) for screening purposes and with Q5 high-fidelity DNA polymerase (New England Biolabs) for cloning purposes. The master mixture for Gibson assembly was prepared as described previously.41 Oligonucleotides and other modification enzymes were purchased from Invitrogen. All primers and oligonucleotides used in this study are listed in Supplementary Table S2. DNA Sequencing and Analysis. DNA was sequenced with an ABI 3730xl analyzer at the Plateforme de Séquençage et 1356


Research Article


fragment (Figure 4). For the point mutation and the insertion, PCR products were sequenced and aligned to confirm the mutations in the phage genome. Mutant phages p2Δ24, p2Δ47Δ24, p2Δ47Δ24Δ42, and p2Δ8% were analyzed by PCR with primers p2_del.24_F and p2_del.24_R and further agarose gel electrophoresis to detect the deletion in orf 24. Amplification on the wild-type phage generated a 738 bp fragment, and amplification on the mutant phages generated a 621 bp fragment (Figure 4). To detect mutations in orf42, primers p2.38 and CB13.24 were used. Amplification on the genome of p2 resulted in an 899 bp fragment, while amplification on mutant phages p2Δ42, p2Δ47Δ24Δ42, and p2Δ8% resulted in an 827 bp fragment (Figure 4). Likewise, to detect mutations in orf49, phage genomes were analyzed by PCR with primers orf48_1−1 and orf49_pNZ123_R. Amplification on the genome of p2 resulted in a 1162 bp fragment and amplification on recombinant phages p2Δ49 and p2Δ8% resulted in a 1032 bp fragment (Figure 4).

PCR products obtained following amplification with primers crRNA_S.pyo_R and Cas9_S.pyo_F6. Construction of Homologous Recombination Templates. All recombination templates were constructed in the shuttle vector pNZ123 digested with XbaI using Gibson assembly (Figure 2). Using the genome of phage p2 as a template, a 547 bp fragment was amplified with primers orf47_pNZ123_F and KO_orf47_R, and a 352 bp fragment was amplified with primers KO_orf47_F and orf47_pNZ123_R. The external primers had complementary ends for assembly at the XbaI restriction site of pNZ123, while the inner primers had complementary ends for annealing. The assembly of the PCR products and linearized vector resulted in pKO47, a template containing a truncated orf47 gene flanked with homologous arms for recombination with the rest of the phage genome (Figure 2A). The same strategy was used to construct p47G86C. A 609 bp fragment was amplified from the phage genome with primers orf47_pNZ123_F and orf47_G86C_R, and a 366 bp fragment was amplified with p rim ers o rf47_G 86C_F and orf47_pNZ123_R. To generate the mutation G86C in orf47, inner primers were designed to contain the appropriate mismatch (Figure 2B). To construct the recombination template pHis47G86C, PCR was performed using p47G86C as the template, rather than the phage genome, since p47G86C already had the desired point mutation. A 527 bp fragment was amplified with primers orf47_pNZ123_F and orf47_6His_R, and a 452 bp fragment was amplified with primers orf47_6His_F and orf47_pNZ123_R. This time, the inner primers had an 18 bp overhang corresponding to a polyhistidine-tag (Figure 2C). Recombination templates pKO24, pKO42, and pKO49 were similarly constructed. For pKO24, a 333 bp fragment was amplified with primers orf24_pNZ123_F and KO_orf24_R, and a 294 bp fragment was amplified with primers KO_orf24_F and orf24_pNZ123_R. The assembly of the PCR products with the linearized pNZ123 generated pKO24, a template containing a truncated orf 24 and suited for homologous recombination with the genome of phage p2. For pKO42, primers orf42_pNZ123_F and KO_orf42_R were used to amplify a 290 bp fragment, and primers KO_orf42_F and orf42_pNZ123_R were used to amplify a 555 bp fragment. The assembly of those fragments with the linearized vector generated pKO42, a recombination template containing a truncated orf42. For pKO49, primers orf49_pNZ123_F and KO_orf49_R were used for the amplification of a 403 bp fragment, and primers KO_orf49_F and orf24_pNZ123_R for the amplification of a 511 bp fragment. The assembly of those fragments with the linearized vector generated pKO49, a recombination template containing a truncated orf49. Following Gibson assembly, the recombination templates were transformed into L. lactis MG1363 and transformants were plated on GM17 Cm 5. The sequences of the inserts in pNZ123 were confirmed by colony PCR with the primers pNZins_F and pNZins_R and subsequent sequencing. Analysis of Recombinant Phages. Isolated phage plaques were analyzed by PCR with pairs of primers amplifying the modified region of the phage genome, and absent from the recombination templates. To detect mutations in orf47, primers CB13.42 and orf48_1−2 were used. For the deletion, amplification on the wild-type phage generated a 738 bp fragment, while amplification on recombinant phages p2Δ47, p2Δ47Δ24, p2Δ47Δ24Δ42, and p2Δ8% generated a 662 bp

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00388. Table S1: Bacterial strains, phages and plasmids used in this study; Table S2: Primers and oligonucleotides used in this study; Table S3: orfs of the virulent phage p2 and their annotations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 418 656 3712. Fax: +1 418 656 2861. E-mail: Sylvain. [email protected]. ORCID

Marie-Laurence Lemay: 0000-0001-8509-8972 Sylvain Moineau: 0000-0002-2832-5101 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Luciano Marraffini for plasmid pCas9. We would like to thank Barbara-Ann Conway (Medical Writer & Editor) for editorial assistance. This work was funded by a team grant from the FRQNT and the Natural Sciences and Engineering Research Council of Canada (Discovery Program). M.-L.L. is supported by scholarships from the Fonds de Recherche du Québec-Nature et Technologies (FRQNT), Novalait and Op +Lait. S.M. holds a T1 Canada Research Chair in Bacteriophages.

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REFERENCES

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Genome Engineering of Virulent Lactococcal Phages Using CRISPR

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