Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9

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.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10

“…The appearance of CEMs was observed at varying frequencies when Types I and II CRISPR-Cas systems were used to edit phages. 27–30 In contrast, neither a PAM nor a seed sequence has been identified for CRISPR-Cas10. 40,45 This system is also extremely tolerant to mismatches between the crRNA and protospacer during antiphage immunity.…”

“…26 However, recent reports have shown that CRISPR-Cas (Clustered regularly-interspaced short palindromic repeats-CRISPR-associated) systems in distinct bacteria can facilitate phage editing. 27–30 CRISPR-Cas systems are a diverse class of prokaryotic immune systems that use small CRISPR RNAs (crRNAs) and Cas nucleases to detect and destroy phages and other nucleic acid invaders. 31–36 In these systems, CRISPR loci maintain an archive of short (30–40 nucleotide) invader-derived sequences called “spacers” integrated between similarly sized DNA repeats.…”

“…CRISPR-Cas systems are remarkably diverse, with two broad Classes and six Types (I–VI) currently described. 37,38 Types I and II CRISPR-Cas systems have recently been used in conjunction with homologous recombination to facilitate phage editing; 27–30 however, the general applicability of this approach in other organisms using distinct CRISPR-Cas systems remains unknown.…”

Comparison of CRISPR and marker based methods for the engineering of phage T7

“…A number of phages have been modified with type II CRISPR systems; including phage 2972 infecting Streptococcus thermophiles, phage P2 infecting Lactococcus. lactis, phiKpS2 infecting Klebsiella pneumoniae and phages T2, T4, T7 and KF1 that infect E. coli respectively [25,[27][28][29][30][31]. A type III CRISPR system has also been used to engineer phages that infect Staphylococcus aureus and Staphylococcus epidermidis [32].…”

Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

“…Methods for genetically engineering phages have recently been reviewed (Pires et al 2016) and commonly involve homologous recombination and recombineering. While such approaches can be improved by clever incorporation of CRISPR-Cas systems (Kiro et al 2014, Lemay et al 2017, Schilling et al 2018, the overall modest efficiencies largely limits mutagenesis to the generation and single-site mutation of chimeric genomes Fane 2015, Doore et al 2017), gene deletions (Uchiyama et al 2009), and limited multi-site mutagenesis (Stockdale et al 2015). A recent approach combines mutagenesis by PCR with degenerate (NNK) primers with in-cell recombination to create variation at specific residues in the tail fiber of T3 phage (Yehl et al 2019).…”