Editing of Phage Genomes—Recombineering-assisted SpCas9 Modification of Model Coliphages T7, T5, and T3

Abstract

Bacteriophages—viruses that infect bacterial cells—are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.

Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis

“…We constructed a T3 mutant lacking the 0 . 3 gene (see Methods and reference 37 ) and tested it for the ability to infect BREX+ cells. Similar to the wild-type T7, wild-type T3 produced plaques on a BREX+ lawn, though the titer was decreased by an order of magnitude and the plaques were smaller than those formed on the BREX-lawn.…”

“…3 gene from T3 genome was achieved using λ RED-assisted SpCas9 genome editing. 37 In short, a plasmid pKDsgRNA was designed to express a spacer against T3 0 . 3 gene.…”

“…Deletion of the 0.3 gene from T3 genome was achieved using λ RED-assisted SpCas9 genome editing. 37 In short, a plasmid pKDsgRNA was designed to express a spacer against T3 0.3 gene. pBAD vector for homologous recombination was provided that carried an insert with 300-bp 5' and 3' fragments bordering the region of desired deletion.…”

Increasing CRISPR/Cas9-mediated gene editing efficiency in T7 phage by reducing the escape rate based on insight into the survival mechanism

“…CRISPR/Cas is an adaptive immune system discovered in bacteria and archaea, and 6 types of CRISPR/Cas systems have been identified. Several CRISPR/Cas-based genome editing methods have been reported for bacteriophages, including the CRISPR/ Cas9 system (type II), the CRISPR/Cas3 system (type I), and the CRISPR/Cas13a system (type VI) [8][9][10][11][12][13]. However, in practical operation, bacteriophages can escape the cleavage of CRISPR/Cas, and the escaped phages are called escapers.…”

“…However, in practical operation, bacteriophages can escape the cleavage of CRISPR/Cas, and the escaped phages are called escapers. The escaper not only reduces the efficiency of bacteriophage genome editing systems but also increases the difficulty of screening positive phages [8][9][10][11]14]. Thus, understanding the escape mechanism of bacteriophages from the cleavage of CRISPR/Cas can advance the development of efficient genome editing tools and accelerate the construction of bacteriophages for therapeutic purposes.…”