Genome Engineering of Virulent Lactococcal Phages Using CRISPR

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Genome engineering of virulent lactococcal phages using CRISPR-Cas9 Marie-Laurence Lemay, Denise M. Tremblay, and Sylvain Moineau ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00388 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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ACS Synthetic Biology

Lemay et al. 1 2

Genome engineering of virulent lactococcal phages

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using CRISPR-Cas9

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Marie-Laurence Lemay1, Denise M. Tremblay1 and Sylvain Moineau1*

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1

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génie, Félix d'Hérelle Reference Center for Bacterial Viruses, and Groupe de recherche en

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écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, QC, G1V 0A6,

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Département de biochimie, de microbiologie, et de bioinformatique, Faculté des sciences et de

Canada

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* To whom correspondence should be addressed. Tel: +1 418 656 3712; Fax: +1 418 656 2861;

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Email: [email protected]

ACS Paragon Plus Environment

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Lemay et al. 14

ABSTRACT

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Phages are biological entities found in every ecosystem. Although much has been learned

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about them in past decades, significant knowledge gaps remain. Manipulating virulent phage

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genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent

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lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in

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various milk fermentation processes and its phage sensitivity poses a constant risk to the cheese

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industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages.

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Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9

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genome editing technology, which is derived from a natural prokaryotic defense mechanism,

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offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-

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Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage.

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Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis

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MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to

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generate precise mutations that allow the study of lytic phage genes and their encoded proteins in

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vivo is described.

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KEYWORDS: virulent bacteriophages, lactic acid bacteria, genome engineering, CRISPR-Cas9,

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homologous recombination, double-strand DNA break repair


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Lemay et al. 32

INTRODUCTION

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In the era of metagenomics, the availability of viral sequence data is continuously

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increasing. Currently, over 2.79 million genes code for viral proteins, but 75% of these have no

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known function and no homolog in public databases1–5. The increasing gap between the number

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of viral genes and the assignment of gene function is mainly due to the abundance of viruses6,

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their diversity7 and difficulties in studying them. Annotations of structural proteins greatly

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outnumber those of non-structural proteins because they are more conserved and easier to study

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with techniques such as SDS-PAGE, mass spectrometry, and electron microscopy8.

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Viruses are obligate parasites as they depend on the host cellular machinery for their

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multiplication. A viral genome can be modified and studied in vivo only while inside a host,

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complicating functional

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(bacteriophages or phages) is also a challenge since their genome never integrates into the

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bacterial chromosome. Moreover, the tight timeframe to engineer virulent phages is dictated by

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the short duration of the infection cycle. The lack of tools to efficiently edit the genome of

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virulent phages partly explains why many non-structural proteins are still uncharacterized9–12.

studies.

Genome

manipulation

of virulent

bacterial

viruses

47

Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and

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CRISPR-associated proteins (CRISPR-Cas) have proven useful for genetically engineering a

49

plethora of organisms (reviewed in

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defense mechanisms found in many prokaryotes that provide sequence-specific protection against

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invasion by foreign nucleic acids14,15. This immune response depends on complementary base

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pairing between the spacer in the CRISPR array (the effective form being a crRNA) and a

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matching protospacer in the genome of the invader14,16–18. CRISPR-Cas systems are extremely

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diverse in nature and are classified according to their associated Cas proteins19. Class 1 systems

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require the activity of a multi-protein complex whereas class 2 systems have a single protein

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effector. The on-target cleavage provided by both classes has been repurposed for genome

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engineering. As such, class 1 subtype I-E CRISPR-Cas systems from E. coli and Vibrio cholerae

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have been used to modify lytic phage genomes9,11. Similarly, the endogenous class 2 subtype II-A

59

system from Streptococcus thermophilus was used to modify the genome of a virulent phage10.

60

13

), including phages9–11. CRISPR-Cas systems are natural

The Cas9 protein is the signature nuclease of the type II systems (class 2)

19,20

. Type II

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systems have in common the need of a protospacer adjacent motif (PAM) next to the target

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site15,21,22. The PAM is specific to every Cas9 ortholog. Streptococcus pyogenes Cas9 (SpCas9),


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Lemay et al. 63

the most intensely studied Cas protein, requires a 5’-NRG-3’ (where R = G or A) PAM to target a

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sequence23,24. SpCas9 can be programmed with a crRNA containing a desired spacer to dictate

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target specificity25. It can target virtually any DNA sequence and the only requirements are PAM

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recognition and a designed crRNA. SpCas9 has been used in numerous organisms (reviewed in

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13

), but it has never been exploited for phage engineering.

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Lactococcal phages are natural inhabitants of milk and because they are mostly unaffected

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by pasteurisation, they pose a risk to milk fermentation processes through infection of industrial

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strains of L. lactis. Virulent lactococcal phages belonging to the sk1 group are by far the most

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prevalent in the dairy industry26,27. Phage p2 is the model for this group and it infects the

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plasmid-cured strain L. lactis MG1363, itself a model Gram-positive bacterial strain for basic

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research. Phage p2 belongs to the Siphoviridae family of the Caudovirales order28. Its 69 nm

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diameter capsid contains a dsDNA genome of 27,595 bp and 49 predicted open reading frames

75

(orfs). While significant research has been done on the structure of phage p2

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from being well understood - almost half of its genes encode proteins with no assigned function.

29,30

, it is still far

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As a model bacterium, L. lactis is amenable to genetic manipulation and efficient means

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are available for introducing foreign DNA into most strains. Many expression systems have also

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been developed to produce recombinant proteins in this species. Yet, it is still not possible to

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readily manipulate the phages of this host. The use of CRISPR-Cas9 for genome engineering has

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only been described in two lactic acid bacteria (LAB): the probiotic Lactobacillus reuteri31 and

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the starter culture S. thermophilus10,14,15,32. CRISPR-mediated immunity to phages was first

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demonstrated in S. thermophilus14. Aside from a plasmid-encoded type III system, no CRISPR-

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Cas system was identified in L. lactis33.

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In this study, CRISPR-Cas9 technology is introduced into L. lactis. The S. pyogenes type

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II-A CRISPR-Cas system is demonstrated to function in L. lactis, efficiently editing the

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previously intractable genome of virulent phage p2. More precisely, targeted gene perturbation,

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single base modification and insertion into orf47, which encodes a conserved non-structural

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protein, was achieved. Although orf47 is part of the sk1 group core-genome, it has no known

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function34. The reproducibility of this method was demonstrated by targeting and disturbing

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additional p2 genes: orf24, orf42 and orf49. The CRISPR-Cas9 tool presented here has the

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potential to significantly expand our knowledge of phage-host interactions by providing new

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information about their many uncharacterized proteins.


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Lemay et al. 94

RESULTS AND DISCUSSION

95 96

Introduction of CRISPR-Cas9 into L. lactis MG1363

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Plasmid pCas9 carries the gene encoding the Cas9 endonuclease as well as the genetic

98

elements needed to generate the tracrRNA and a crRNA. These genetic elements were all derived

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from the type II-A CRISPR-Cas system of S. pyogenes SF37024. Since pCas9 does not replicate

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in L. lactis, the variant pNZCas9 was generated by cloning the above CRISPR-Cas9 system into

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the broad-host-range, high-copy number plasmid, pNZ12335, and transforming it into E. coli

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NEB5α (host for cloning). The integrity of pNZCas9 was confirmed by sequencing. The plasmid

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was then transformed into L. lactis MG1363. All transformation attempts were unsuccessful, as

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all colonies tested had lost the CRISPR-Cas9 components. These results suggested that Cas9 may

105

be toxic to L. lactis when expressed from a high-copy vector. Decreasing the expression of Cas9

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was likely necessary.

107

Another derivative of pCas9 was constructed using the low-copy number plasmid,

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pTRKL236. The resulting pL2Cas9 was successfully transformed into E. coli and then into L.

109

lactis MG1363. The plasmid was sequenced in its entirety and no mutations were found.

110 111

Efficiency of pNZCas9 and pL2Cas9 protection against phage infection

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Because pNZCas9 was successfully transformed into E. coli NEB5α, its efficiency in

113

protecting E. coli against lytic phage infection could be tested. A spacer targeting the gene coding

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for the phage T4 DNA replication protein repEA was cloned into the crRNA of pNZCas9 to

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generate pNZCas9-T4, which was introduced into E. coli MG1655. Colonies were selected

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randomly and the construction confirmed by sequencing. The level of phage resistance

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(interference) provided by pNZCas9-T4 was measured by efficiency of plaquing (EOP). The

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EOP was calculated by dividing the phage titer obtained on the resistant strain (E. coli MG1655

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pNZCas9-T4) by the phage titer obtained on the sensitive strain (E. coli MG1655 pNZCas9).

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Following infection of the two bacterial strains with virulent phage T4 (biological triplicates), a

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modest reduction in EOP of 0.71 ± 0.01 was observed.

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Similarly, the phage resistance provided by pL2Cas9 was investigated in L. lactis. SpCas9

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was programmed to cleave a specific region of phage p2 orf47 by cloning a new spacer into the

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crRNA of pL2Cas9 to generate pL2Cas9-47 (Figure 1). The ability of pL2Cas9-47 to protect L.


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Lemay et al. 125

lactis MG1363 against infection by virulent phage p2 was then tested. The assays were

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performed at three temperatures: 30°C, the optimal growth temperature for L. lactis, 37°C, the

127

optimal growth temperature for S. pyogenes and 35°C, an intermediate temperature. In all cases,

128

the reduction in EOP was modest but sufficient to show that the S. pyogenes CRISPR-Cas9

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system can interfere with phage infection in L. lactis MG1363. EOPs of 0.49 ± 0.13, 0.31 ± 0.14,

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and 0.44 ± 0.03 were observed after incubation at 30°C, 35°C and 37°C, respectively.

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Sequence analysis of escaping phages

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After infecting the transformant L. lactis MG1363 pL2Cas9-47 with phage p2 at 30°C, 92

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phage plaques were picked and the phage genomic region targeted by CRISPR-Cas9 was

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analyzed using PCR (primers CB13.42 and orf48_1-2) and subsequent sequencing. No mutations

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were detected in either the protospacer or the PAM of these seemingly CRISPR-Escaping

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Mutants (CEMs). These results were likely due to the weak phage resistance phenotype conferred

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by pL2Cas9-47 and the plaques were not from CEMs (phages with a mutated protospacer or

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PAM) but from wild-type phage p2.

140 141

Genome engineering of the virulent phage p2

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To determine if the interference provided by the S. pyogenes CRISPR-Cas9 system in L.

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lactis was sufficient to readily modify the genome of the virulent phage p2, templates for

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homologous recombination were provided to facilitate screening for specific mutations. These

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templates were designed to contain not only a desired mutation and homologous flanking arms to

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allow recombination, but also to lack the motif targeted by SpCas9 and to prevent further

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interference by the system (Figure 2). Gene deletions, point mutation within a gene and insertion

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of desired nucleotides were successfully achieved, all within the genome of the virulent

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lactococcal phage p2 using pL2Cas9 (Figure 3).

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Deletion

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To create a deletion in orf47 of phage p2, a recombination template was constructed by

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Gibson assembly (see Methods) and cloned into pNZ123 (in L. lactis) to generate pKO47 (Figure

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2A). Of note, the plasmid pNZ123 is compatible with pTRKL2, the vector backbone of pL2Cas9.

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After confirmation of pKO47 by sequencing, the adapted gene editing tool pL2Cas9-47 was


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Lemay et al. 156

transformed into L. lactis MG1363 containing pKO47. The resulting strain (L. lactis MG1363

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pKO47 pL2Cas9-47) was then challenged with increasing dilutions of phage p2 in order to obtain

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distinct lysis plaques. Three randomly selected phage plaques were analyzed by PCR. Two

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plaques showed two distinct bands corresponding to a mixed population of wild-type and

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recombinant phages (data not shown). The third plaque showed a single band corresponding to

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the expected deletion of 76 bp in orf47 (Figure 4). Still, we assumed that this third plaque also

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contained a mixed population of phages, but with a higher proportion of recombinant phages, as

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readily detected by a shorter PCR product. Therefore, the recombinant phage was purified by

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three sequential rounds of infection on the same strain. This methodology ensured the removal of

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any remaining wild-type phages.

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The deletion in orf47 resulted in a truncated protein retaining only eight N-terminal amino

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acids (out of 43) from Orf47. Full genome sequencing of the phage mutant p2∆47 detected no

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additional mutations, indicating no genetic compensation and, more importantly, no off-target

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activity with this CRISPR-Cas9 adapted tool. Disrupting this gene indicated that it is a bona fide

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non-essential gene for phage p2, under the conditions tested. This phage mutant will allow

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investigation of the role of Orf47 during the phage infection process.

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Taken altogether, it is possible to recover a recombinant phage despite the modest phage

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resistance phenotype provided by pL2Cas9-47. Of note, a similar experiment was performed with

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phage p2 and L. lactis MG1363 containing only the recombination template pKO47. No

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recombinant phage was obtained (out of the 45 phage plaques tested), highlighting the

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requirement for pL2Cas9-47 to efficiently obtain the desired phage mutant.

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Point mutation

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Using the same strategy as above, the recombination template p47G86C (Figure 2B) was

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constructed to generate a point mutation within orf47. The point mutation G86C changed the 5’-

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TGG-3’ PAM to 5’-TGC-3’, preventing SpCas9 from targeting a recombinant phage. L. lactis

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MG1363 containing both p47G86C and pL2Cas9-47 was infected with phage p2 and a randomly

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selected phage plaque was purified thrice on the same host to obtain phage p247G86C. The

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mutation in the recombinant phage genome was confirmed by sequencing the PCR product of

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orf47. This point mutation showed that a single mutation in the PAM was sufficient for the

186

recombinant p247G86C to bypass the interference activity of CRISPR-Cas9.


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Lemay et al. 187

Insertion

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Another recombination template (pHis47G86C) was constructed to introduce an 18 bp

189

sequence corresponding to a polyhistidine-tag at the N-terminus of Orf47, as well as the point

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mutation G86C (Figure 2C), described above. The mutation changed amino acid 29 from glycine

191

to alanine, which is unlikely to change the activity of Orf47 (data not shown). L. lactis MG1363

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containing pHis47G86C and pL2Cas9-47 was then challenged with the virulent phage p2 to

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engineer the recombinant phage p2His47G86C. After the first round of infection, one randomly

194

selected phage was plaque purified three times using a bacterial strain carrying only pL2Cas9-47.

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The presence of the recombination template during purification of p2His47G86C caused loss of

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the His-tag, but purification with L. lactis MG1363 pL2Cas9-47 addressed this problem. Both the

197

18 bp insertion and the point mutation in recombinant phage p2His47G86C were confirmed by

198

sequencing.

199 200

Targeting other phage p2 genes

201

To evaluate the robustness of pL2Cas9 for modifying the genome of the virulent phage

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p2, additional mutations were generated. Using the same strategy as described for pL2Cas9-47

203

(Figure 1), new spacers were cloned into the crRNA of pL2Cas9 to construct pL2Cas9-24,

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pL2Cas9-42 and pL2Cas9-49 to target orf24, orf42 and orf49, respectively. To delete these three

205

genes of unknown function, recombination templates pKO24, pKO42 and pKO49 were

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constructed using the same method as for pKO47 (Figure 2A). All recombination templates and

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crRNA were confirmed by sequencing.

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L. lactis MG1363 pKO24 pL2Cas9-24, L. lactis MG1363 pKO42 pL2Cas9-42 and L.

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lactis MG1363 pKO49 pL2Cas9-49 were independently infected with phage p2. One randomly

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selected phage plaque was selected for each bacterial strain. Mutant phages p2∆24, p2∆42 and

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p2∆49 were each purified by three rounds of infection on their cognate host. Deletions were

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analyzed by PCR and subsequent migration on a 2% agarose gel (Figure 4). PCR products were

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also sequenced to confirm the mutations. No apparent differences in the efficiency of mutation

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were observed since all phage recombinants were successfully obtained using the same strategy.

215

The 117 bp deletion in virulent phage p2 early-expressed gene orf24 resulted in a

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truncated protein retaining 26 N-terminal and 13 C-terminal amino acids (out of 78) from Orf24.

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Full genome sequencing of the phage mutant p2∆24 showed no other mutation. The 72 bp


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Lemay et al. 218

deletion in the early-expressed gene orf42 resulted in a truncated protein retaining 7 N-terminal

219

and 27 C-terminal amino acids (out of 60) from Orf42. The 132 bp deletion in the middle-

220

expressed gene orf49 resulted in a truncated protein retaining only 12 N-terminal amino acids

221

(out of 55) from Orf49.

222 223

Sequential mutations

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A mutant of phage p2 carrying deletions in multiple genes was generated by sequential

225

recombination coupled with the selective pressure conferred by CRISPR-Cas9. L. lactis MG1363

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pKO24 pL2Cas9-24 was first infected with the recombinant phage p2∆47. The sequence of the

227

resulting recombinant phage p2∆47∆24 was confirmed by full genome sequencing. To obtain the

228

mutant p2∆47∆24∆42, L. lactis MG1363 pKO42 pL2Cas9-42 was then infected with phage

229

p2∆47∆24. The three deletions in the genome of the resulting phage were confirmed by PCR,

230

agarose gel electrophoresis (Figure 4) and sequencing. L. lactis MG1363 pKO49 pL2Cas9-49

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was then infected with p2∆47∆24∆42 to obtain a derivative of phage p2 lacking four genes out of

232

the 49 annotated ones (8%). The four deletions in phage p2∆8% were also confirmed by PCR, gel

233

electrophoresis (Figure 4) and sequencing. Knockouts of those four genes suggest that they are

234

non-essential under the conditions used.

235 236

Conclusion

237

This study shows that the programmable on-target cleavage provided by SpCas9 can be

238

used to reliably generate precise mutations in the genome of the lactococcal virulent phage p2.

239

Although naturally occurring homologous recombination can be co-opted to generate mutations

240

in a phage genome, recombination rates are very low and the selection of a specific mutant can be

241

time-consuming. The method described here is simple to carry out and highly efficient, thereby

242

avoiding large-scale plaque screening to obtain the desired recombinant virulent phage. In all

243

cases, we observed equal mutation efficiencies. While not tested here, it is very likely that Cas9

244

variants could also be used in L. lactis, including the catalytically inactive Cas9, to modulate

245

gene expression37,38. A total of 4992 PAMs (NRG) are spread across the genome of phage p2 and

246

each orf can be targeted by SpCas9, offering the possibility of studying the functions of these orfs

247

in vivo. Knockout studies are an efficient way to begin elucidating protein function, but they are

248

not suitable for investigating genes essential for phage multiplication. To study the role of


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Lemay et al. 249

essential genes in vivo, non-disruptive mutations must be generated. Being able to fuse a His-tag

250

to a phage protein of interest provides a new tool to purify that protein and identify its binding

251

partners during the phage infection process. Finally, pTRKL2, the vector backbone of pL2Cas9,

252

is stable in many other LAB36, providing opportunities to edit the genomes of other phages

253

infecting these industrially relevant bacterial strains.

254 255

METHODS

256 257

Bacterial strains, phages and growth conditions

258

All bacterial strains, phages and plasmids used in this study are listed in Supplementary

259

Table S1. Phages p2 (GenBank NC_009004.1) and T4 as well as their hosts were obtained from

260

the Félix d’Hérelle Reference Center for Bacterial Viruses (www.phage.ulaval.ca). The bacterial

261

strain L. lactis MG1363 and its derivatives were grown statically at 30°C in M17 broth (Oxoid,

262

Ontario, Canada) supplemented with 0.5% glucose (GM17), unless otherwise stated. For solid

263

media, agar (1.0% w/v) was added to GM17 broth. For transformation, electrocompetent cells

264

were prepared as described previously39. To avoid plasmid loss, chloramphenicol or

265

erythromycin was added to the media to a final concentration of 5 µg/ml (Cm 5 or Em 5). E. coli

266

strains were grown in LB medium or BHI and incubated at 37°C with agitation. For cloning

267

purposes, chemically competent E. coli NEB5α were purchased (New England Biolabs). When

268

needed, chloramphenicol was added to a final concentration of 20 µg/ml (Cm 20) in LB and

269

erythromycin was added to a final concentration of 150 µg/ml (Em 150) in BHI. For solid media,

270

agar (1.5% w/v) was added to LB or BHI broth. For phage infection, media were supplemented

271

with 10 mM CaCl2 and no antibiotic was added. For double layer plaque assays40, plates

272

contained a bottom layer of the appropriate medium supplemented with 1.0% (w/v) agar and a

273

top layer of the medium supplemented with 0.75% (w/v) agar.

274 275

Reagents and enzymes

276

Plasmids were purified from overnight bacterial cultures using the QIAprep Spin

277

Miniprep kit (Qiagen). Prior to plasmid extraction, L. lactis cultures were treated with lysozyme

278

(30 mg/ml, 30 minutes, 37˚C). Restriction enzymes were purchased from New England Biolabs.

279

Polymerase chain reactions (PCR) were performed with Taq polymerase (Feldan) for screening


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Lemay et al. 280

purposes and with Q5 high-fidelity DNA polymerase (New England Biolabs) for cloning

281

purposes. The master mixture for Gibson assembly was prepared as described previously41.

282

Oligonucleotides and other modification enzymes were purchased from Invitrogen. All primers

283

and oligonucleotides used in this study are listed in Supplementary Table S2.

284 285

DNA sequencing and analysis

286

DNA was sequenced with an ABI 3730xl analyzer at the Plateforme de Séquençage et de

287

Génotypage des Génomes at the CHUL center. For whole-genome and complete plasmid

288

sequencing, libraries were prepared using the Nextera XT DNA library preparation kit (Illumina)

289

according to the manufacturer's instructions. Sequencing was performed on a MiSeq system

290

using a MiSeq reagent kit v2 (Illumina). The sequences were analyzed using Geneious 7.1.4.

291 292

Construction of pNZCas9

293

Plasmid pCas924 was a gift from Luciano Marraffini (Addgene plasmid # 42876). It was

294

digested with SalI and XbaI, generating two fragments of 3524 bp and 5802 bp, the latter

295

retaining the CRISPR-Cas9 components. Plasmid pNZ123 was amplified with the primers

296

pNZ123_Gibson_F and pNZ123_Gibson_R. Both primers were designed to have overhangs

297

suited for Gibson assembly41 with the appropriate pCas9 fragment. Prior to Gibson assembly, the

298

two fragments to be assembled (5802 bp and PCR product of pNZ123) were purified using the

299

QIAquick PCR purification kit (Qiagen). The assembled product was transformed into E. coli

300

NEB5α and transformants were isolated on LB Cm 20 plates. Three individual colonies were

301

screened for the presence of pNZCas9. They were grown overnight in fresh media and their

302

plasmids isolated. The plasmids were then digested with BsaI, which has two unique restriction

303

sites within the crRNA on both sides of the spacer. Restricted plasmids were separated on a 2%

304

(w/v) agarose gel to confirm that both fragments had been annealed. The CRISPR-Cas9

305

components were sequenced and aligned with the sequence of pCas9 to verify their integrity.

306 307 308

Construction of pL2Cas9 To

construct

pL2Cas9,

plasmid

pTRKL2

was

amplified

with

the

primers

309

pTRKL2_Gibson_F and pTRKL2_Gibson_R, both located in the lacZ reporter sequence,

310

yielding a 6419 bp fragment. Using the same strategy as described above, pTRKL2 was


11


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Page 12 of 24

Lemay et al. 311

assembled with the CRISPR-Cas9 components of pCas9. The assembled product, named

312

pL2Cas9, was transformed into E. coli NEB5α and transformants were isolated on BHI Em 150

313

plates. Individual colonies were screened for the presence of cas9 by colony PCR using

314

Cas9_S.pyo_F5 and Cas9_S.pyo_R1 primers. Colonies yielding a PCR fragment of the correct

315

size were grown overnight. Plasmids were purified from these cultures and sequenced to confirm

316

the integrity of the CRISPR-Cas9 components.

317 318

New spacer cloning in pNZCas9 and pL2Cas9

319

Both plasmids were digested with BsaI and purified by precipitation with salts and

320

ethanol. Oligonucleotides PS_T4 and PS_T4_RC were designed to match a protospacer located

321

in the gene repEA of phage T4 and to have ends complementary to the BsaI site to allow cloning

322

into pNZCas9. The oligonucleotides were annealed and ligated overnight into the digested

323

plasmid, using an insert:vector molar ratio of 3:1 with approximately 0.1 µg of total DNA. The

324

resulting pNZCas9-T4 was transformed into E. coli MG1655, host of phage T4.

325

Using the same method, oligonucleotides PS_47 and PS_47_RC were annealed and

326

cloned into pL2Cas9. This spacer is complementary to a protospacer flanked by a PAM and

327

located in orf47 of phage p2 (Figure 1). The resulting pL2Cas9-47 was dialysed on a membrane

328

and electroporated into L. lactis MG1363, host of phage p2. Likewise, oligonucleotides PS_24

329

and PS_24_RC, PS_42 and PS_42_RC, as well as PS_49 and PS_49_RC were cloned into the

330

crRNA of pL2Cas9 to generate plasmids pL2Cas9-24, pL2Cas9-42 and pL2Cas9-49, respectively.

331

The presence of the correct spacers was confirmed by sequence analysis of the PCR products

332

obtained following amplification with primers crRNA_S.pyo_R and Cas9_S.pyo_F6.

333 334

Construction of homologous recombination templates

335

All recombination templates were constructed in the shuttle vector pNZ123 digested with

336

XbaI using Gibson assembly (Figure 2). Using the genome of phage p2 as a template, a 547 bp

337

fragment was amplified with primers orf47_pNZ123_F and KO_orf47_R, and a 352 bp fragment

338

was amplified with primers KO_orf47_F and orf47_pNZ123_R. The external primers had

339

complementary ends for assembly at the XbaI restriction site of pNZ123, while the inner primers

340

had complementary ends for annealing. The assembly of the PCR products and linearized vector


12

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Lemay et al. 341

resulted in pKO47, a template containing a truncated orf47 gene flanked with homologous arms

342

for recombination with the rest of the phage genome (Figure 2A).

343

The same strategy was used to construct p47G86C. A 609 bp fragment was amplified

344

from the phage genome with primers orf47_pNZ123_F and orf47_G86C_R, and a 366 bp

345

fragment was amplified with primers orf47_G86C_F and orf47_pNZ123_R. To generate the

346

mutation G86C in orf47, inner primers were designed to contain the appropriate mismatch

347

(Figure 2B).

348

To construct the recombination template pHis47G86C, PCR was performed using

349

p47G86C as the template, rather than the phage genome, since p47G86C already had the desired

350

point mutation. A 527 bp fragment was amplified with primers orf47_pNZ123_F and

351

orf47_6His_R, and a 452 bp fragment was amplified with primers orf47_6His_F and

352

orf47_pNZ123_R. This time, the inner primers had an 18 bp overhang corresponding to a

353

polyhistidine-tag (Figure 2C).

354

Recombination templates pKO24, pKO42 and pKO49 were similarly constructed. For

355

pKO24, a 333 bp fragment was amplified with primers orf24_pNZ123_F and KO_orf24_R, and a

356

294 bp fragment was amplified with primers KO_orf24_F and orf24_pNZ123_R. The assembly

357

of the PCR products with the linearized pNZ123 generated pKO24, a template containing a

358

truncated orf24 and suited for homologous recombination with the genome of phage p2. For

359

pKO42, primers orf42_pNZ123_F and KO_orf42_R were used to amplify a 290 bp fragment, and

360

primers KO_orf42_F and orf42_pNZ123_R were used to amplify a 555 bp fragment. The

361

assembly of those fragments with the linearized vector generated pKO42, a recombination

362

template containing a truncated orf42. For pKO49, primers orf49_pNZ123_F and KO_orf49_R

363

were used for the amplification of a 403 bp fragment, and primers KO_orf49_F and

364

orf24_pNZ123_R for the amplification of a 511 bp fragment. The assembly of those fragments

365

with the linearized vector generated pKO49, a recombination template containing a truncated

366

orf49.

367

Following Gibson assembly, the recombination templates were transformed into L. lactis

368

MG1363 and transformants plated on GM17 Cm 5. The sequences of the inserts in pZN123 were

369

confirmed by colony PCR with the primers pNZins_F and pNZins_R and subsequent sequencing.


13


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Page 14 of 24

Lemay et al. 370

Analysis of recombinant phages

371

Isolated phage plaques were analyzed by PCR with pairs of primers amplifying the

372

modified region of the phage genome, and absent from the recombination templates. To detect

373

mutations in orf47, primers CB13.42 and orf48_1-2 were used. For the deletion, amplification on

374

the wild-type phage generated a 738 bp fragment, while amplification on recombinant phages

375

p2∆47, p2∆47∆24, p2∆47∆24∆42 and p2∆8% generated a 662 bp fragment (Figure 4). For the

376

point mutation and the insertion, PCR products were sequenced and aligned to confirm the

377

mutations in the phage genome. Mutant phages p2∆24, p2∆47∆24, p2∆47∆24∆42 and p2∆8%

378

were analyzed by PCR with primers p2_del.24_F and p2_del.24_R and further agarose gel

379

electrophoresis to detect the deletion in orf24. Amplification on the wild-type phage generated a

380

738 bp fragment, and amplification on the mutant phages generated a 621 bp fragment (Figure 4).

381

To detect mutations in orf42, primers p2.38 and CB13.24 were used. Amplification on the

382

genome of p2 resulted in an 899 bp fragment, while amplification on mutant phages p2∆42,

383

p2∆47∆24∆42 and p2∆8% resulted in an 827 bp fragment (Figure 4). Likewise, to detect

384

mutations in orf49, phage genomes were analyzed by PCR with primers orf48_1-1 and

385

orf49_pNZ123_R. Amplification on the genome of p2 resulted in a 1162 bp fragment and

386

amplification on recombinant phages p2∆49 and p2∆8% resulted in a 1032 bp fragment (Figure

387

4).

388 389 390 391

SUPPORTING INFORMATION 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.

392 393

ACKNOWLEDGEMENTS

394

We thank Luciano Marraffini for plasmid pCas9. We would like to thank Barbara-Ann

395

Conway (Medical Writer & Editor) for editorial assistance. This work was funded by a team

396

grant from the FRQNT and the Natural Sciences and Engineering Research Council of Canada

397

(Discovery program). M.-L.L. is supported by scholarships from the Fonds de Recherche du

398

Québec - Nature et Technologies (FRQNT), Novalait and Op+Lait. S.M. holds a T1 Canada

399

Research Chair in Bacteriophages.


14

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Lemay et al. 400

REFERENCES

401

(1) Paez-Espino, D., Eloe-Fadrosh, E. A., Pavlopoulos, G. A., Thomas, A. D., Huntemann, M.,

402

Mikhailova, N., Rubin, E., Ivanova, N. N., and Kyrpides, N. C. (2016) Uncovering Earth’s

403

virome. Nature 536, 425–430.

404

(2) Edwards, R. A., and Rohwer, F. (2005) Viral metagenomics. Nat. Rev. Microbiol. 3, 504–510.

405

(3) Hurwitz, B. L., U’Ren, J. M., and Youens-Clark, K. (2016) Computational prospecting the

406

great viral unknown. FEMS Microbiol. Lett. 363, fnw077.

407

(4) Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local

408

alignment search tool. J. Mol. Biol. 215, 403–410.

409

(5) Kelley, L. A., and Sternberg, M. J. E. (2009) Protein structure prediction on the Web: a case

410

study using the Phyre server. Nat. Protoc. 4, 363–371.

411

(6) Suttle, C. A. (2005) Viruses in the sea. Nature 437, 356–361.

412

(7) Breitbart, M., and Rohwer, F. (2005) Here a virus, there a virus, everywhere the same virus?

413

Trends Microbiol. 13, 278–284.

414

(8) Fokine, A., and Rossmann, M. G. (2014) Molecular architecture of tailed double-stranded

415

DNA phages. Bacteriophage 4, e28281.

416

(9) Kiro, R., Shitrit, D., and Qimron, U. (2014) Efficient engineering of a bacteriophage genome

417

using the type I-E CRISPR-Cas system. RNA Biol. 11, 42–44.

418

(10) Martel, B., and Moineau, S. (2014) CRISPR-Cas: an efficient tool for genome engineering

419

of virulent bacteriophages. Nucleic Acids Res. 42, 9504–9513.

420

(11) Box, A. M., McGuffie, M. J., O’Hara, B. J., and Seed, K. D. (2015) Functional analysis of

421

bacteriophage immunity through a Type I-E CRISPR-Cas system in Vibrio cholerae and its

422

application in bacteriophage genome engineering. J. Bacteriol. 198, 578–590.

423

(12) Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J., and Lu, T. K. (2016) Genetically

424

engineered phages: a review of advances over the last decade. Microbiol. Mol. Biol. Rev. 80,

425

523–543.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

Lemay et al. 426

(13) Hsu, P. D., Lander, E. S., and Zhang, F. (2014) Development and applications of CRISPR-

427

Cas9 for genome engineering. Cell 157, 1262–1278.

428

(14) Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.

429

A., and Horvath, P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes.

430

Science 315, 1709–1712.

431

(15) Garneau, J. E., Dupuis, M. E., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P.,

432

Fremaux, C., Horvath, P., Magadan, A. H., and Moineau, S. (2010) The CRISPR/Cas bacterial

433

immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71.

434

(16) Mojica, F. J. M., Diez-Villasenor, C., Garcia-Martinez, J., and Soria, E. (2005) Intervening

435

sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol.

436

Evol. 60, 174–182.

437

(17) Pourcel, C., Salvignol, G., and Vergnaud, G. (2005) CRISPR elements in Yersinia pestis

438

acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools

439

for evolutionary studies. Microbiology 151, 653–663.

440

(18) Bolotin, A., Quinquis, B., Sorokin, A., and Ehrlich, S. D. (2005) Clustered regularly

441

interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

442

Microbiology 151, 2551–2561.

443

(19) Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J.,

444

Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J.,

445

Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J.,

446

Backofen, R., and Koonin, E. V. (2015) An updated evolutionary classification of CRISPR-Cas

447

systems. Nat. Rev. Microbiol. 13, 722–736.

448

(20) Esvelt, K. M., Mali, P., Braff, J. L., Moosburner, M., Yaung, S. J., and Church, G. M. (2013)

449

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–

450

1121.

451

(21) Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., Eckert,

452

M. R., Vogel, J., and Charpentier, E. (2011) CRISPR RNA maturation by trans-encoded small


16

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lemay et al. 453

RNA and host factor RNase III. Nature 471, 602–607.

454

(22) Shah, S. A., Erdmann, S., Mojica, F. J. M., and Garrett, R. A. (2013) Protospacer recognition

455

motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899.

456

(23) Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y.,

457

Fine, E. J., Wu, X., Shalem, O., Cradick, T. J., Marraffini, L. A., Bao, G., and Zhang, F. (2013)

458

DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832.

459

(24) Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013) RNA-guided editing

460

of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239.

461

(25) Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012) A

462

programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337,

463

816–821.

464

(26) Deveau, H., Labrie, S. J., Chopin, M. C., and Moineau, S. (2006) Biodiversity and

465

classification of lactococcal phages. Appl. Environ. Microbiol. 72, 4338–4346.

466

(27) Mahony, J., Murphy, J., and van Sinderen, D. (2012) Lactococcal 936-type phages and dairy

467

fermentation problems: from detection to evolution and prevention. Front Microbiol. 3, 335.

468

(28) Ackermann, H.-W. (2007) 5500 phages examined in the electron microscope. Arch. Virol.

469

152, 227–243.

470

(29) Sciara, G., Bebeacua, C., Bron, P., Tremblay, D., Ortiz-Lombardia, M., Lichière, J., van

471

Heel, M., Campanacci, V., Moineau, S., and Cambillau, C. (2010) Structure of lactococcal phage

472

p2 baseplate and its mechanism of activation. P Natl Acad Sci USA 107, 6852–6857.

473

(30) Bebeacua, C., Lai, L., Vegge, C. S., Brondsted, L., van Heel, M., Veesler, D., and Cambillau,

474

C. (2013) Visualizing a complete Siphoviridae member by single-particle electron microscopy:

475

the structure of lactococcal phage TP901-1. J. Virol. 87, 1061–1068.

476

(31) Oh, J.-H., and van Pijkeren, J.-P. (2014) CRISPR-Cas9-assisted recombineering in

477

Lactobacillus reuteri. Nucleic Acids Res. 42, e131.

478

(32) Horvath, P., Romero, D. A., Coûté-Monvoisin, A.-C., Richards, M., Deveau, H., Moineau,


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

Lemay et al. 479

S., Boyaval, P., Fremaux, C., and Barrangou, R. (2008) Diversity, activity, and evolution of

480

CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412.

481

(33) Millen, A. M., Horvath, P., Boyaval, P., and Romero, D. A. (2012) Mobile CRISPR/Cas-

482

mediated bacteriophage resistance in Lactococcus lactis. PLoS One 7, e51663.

483

(34) Rousseau, G. M., and Moineau, S. (2009) Evolution of Lactococcus lactis phages within a

484

cheese factory. Appl. Environ. Microbiol. 75, 5336–5344.

485

(35) De Vos, W. M. (1987) Gene cloning and expression in lactic streptococci. FEMS Microbiol.

486

Lett. 46, 281–295.

487

(36) O’Sullivan, D. J., and Klaenhammer, T. R. (1993) High- and low-copy-number Lactococcus

488

shuttle cloning vectors with features for clone screening. Gene 137, 227–231.

489

(37) Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., and

490

Lim, W. A. (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific

491

control of gene expression. Cell 152, 1173–1183.

492

(38) Larson, M. H., Gilbert, L. A., Wang, X., Lim, W. A., Weissman, J. S., and Qi, L. S. (2013)

493

CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8,

494

2180–2196.

495

(39) Holo, H., and Nes, I. F. (1989) High-frequency transformation, by electroporation, of

496

Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl.

497

Environ. Microbiol. 55, 3119–3123.

498

(40) Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E., and Johnson, R. P. (2009)

499

Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 501,

500

69–76.

501

(41) Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. a, Smith, H. O., Iii, C.

502

A. H., and America, N. (2009) Enzymatic assembly of DNA molecules up to several hundred

503

kilobases. Nat. Methods 6, 343–345.

504


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Graphical abstract


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Page 20 of 24

Lemay et al.

508 509

Figure 1. New spacer cloning in pL2Cas9. (A) Schematic representation of phage p2 genomic

510

organization. The gene expression modules are designated by coloured boxes. Late-expressed

511

phage genes during the infection of L. lactis are enclosed in a red box, while early- and middle-

512

expressed genes are enclosed in green and yellow boxes, respectively. Black arrows indicate

513

genes of unknown function and white arrows indicate genes with assigned function. (B) Orf47 is

514

a putative protein of no known function. (C) The nucleotide sequence of orf47 in the phage p2

515

genome is partially shown. The targeted PAM and protospacer are in black and light grey,

516

respectively. To generate a CRISPR-Cas9-mediated double-stranded break in orf47, the 30 bp

517

corresponding to the protospacer were cloned as a spacer between two repeats (diamonds) in the

518

crRNA of pL2Cas9.


20

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Lemay et al.

519 520 521

Figure 2. Construction of three homologous recombination templates. (A) To construct

522

pKO47, a 547 bp fragment (left, containing the 3’-end of orf45, orf46 and the 5’-end of orf47)

523

and a 352 bp fragment (right, containing the 3’-end of orf47 and the 5’-end of orf48) were

524

amplified from the phage p2 genome. The external primers had overlaps (green and blue)

525

appropriate for inserting the amplicons into linearized vector using Gibson assembly. The inner

526

primers had complementary overhangs (pink) for annealing. Assembly of the PCR products

527

disrupted the sequence of orf47 (hatched gene). The protospacer (grey) and the PAM (black) are


21


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Page 22 of 24

Lemay et al. 528

absent from pKO47, preventing CRISPR-Cas9 interference with the generated recombinant

529

phages. The same strategy was used to construct pKO24, pKO42 and pKO49. (B) To construct

530

p47G86C, a 609 bp fragment (left, containing the 3’-end orf45, orf46 and the 5’-end of orf47)

531

and a 366 bp fragment (right, containing the 3’-end of orf47 and the 5’-end of orf48) were

532

amplified from the phage p2 genome using the same external primers. The inner primers were

533

designed to generate a point mutation (*) removing the PAM from the sequence, thereby

534

preventing CRISPR-Cas9 interference. (C) To construct pHis47G86C, PCR reactions were

535

performed using p47G86C as the template, rather than the phage genome, since p47G86C already

536

had the point mutation to eliminate the PAM. The inner primers had 18 bp overhangs (yellow) to

537

allow fusion of an N-terminal polyhistitine-tag immediately after the start codon.


22

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Lemay et al.

538 539 540

Figure 3. Targeted genome editing of virulent phage p2. (1) L. lactis MG1363 containing the

541

plasmids pKO47 and pL2Cas9-47 was infected with virulent phage p2. (2) Shortly after the viral

542

DNA enters the cell, the CRISPR-Cas9 system recognizes the targeted protospacer (grey) and

543

PAM (black) located in orf47 and generates a double-stranded break. (3) The lesion is then

544

repaired with the recombination template pKO47, to permanently inactivate orf47. (4) The

545

resulting edited genome lacks the target sequence, therefore avoiding cleavage by the CRISPR-

546

Cas9 system. (5) The recombinant phage p2∆47 is released through cell lysis. The same principle

547

applies for all the genome modifications described, the only difference being the recombination

548

template used to repair the lesion.

549


23


Lemay et al.

8

9

10

11

∆8%

∆8%

7

∆47/24/42

6

∆49

5

p2

4

∆47/24/42

17

orf47

18

19

20

21 ∆8%

16

p2

15

∆8%

14

∆47/24/42

13

orf42

∆24/47

12

∆47

orf24

∆49

3

∆24/47

2

∆24

1

p2

550

p2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

orf49

551 552

Figure 4. PCR-based confirmation of deletions in the genome of phage p2 recombinants.

553

PCR products were migrated on a 2% agarose gel, together with 1 kb plus DNA ladder (Life

554

Technologies) (lanes 1, 7, 12 and 18). The deletion in orf24 was confirmed by the shorter PCR

555

products obtained with mutated phages p2∆24, p2∆47∆24, p2∆47∆24∆42 and p2∆8% (lanes 3 to

556

6, respectively), compared to the wild-type phage p2 (lane 2). The deletion in orf42 was

557

confirmed by the shorter PCR products obtained with mutated phages p2∆42, p2∆47∆24∆42 and

558

p2∆8% (lanes 9 to 11, respectively), compared to the wild-type phage p2 (lane 8). The deletion in

559

orf47 was confirmed by the shorter PCR products obtained with mutated phages p2∆47,

560

p2∆47∆24, p2∆47∆24∆42 and p2∆8% (lanes 14 to 17, respectively), compared to the wild-type

561

phage p2 (lane 13). The deletion in orf49 was confirmed by the shorter PCR products obtained

562

with mutated phages p2∆49 and p2∆8% (lanes 20 and 21, respectively), compared to the wild-

563

type phage p2 (lane 19).


24

Genome Engineering of Virulent Lactococcal Phages Using CRISPR

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