Repurposing an Endogenous CRISPR-Cas System to Generate and Study Subtle Mutations in Bacteriophages

CRISPR J. 2024 Dec;7(6):343-354. doi: 10.1089/crispr.2024.0047. Epub 2024 Sep 30.

Abstract

While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in Pectobacterium carotovorum phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.

MeSH terms

  • Bacteriophages* / genetics
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Gene Editing* / methods
  • Genome, Viral
  • Homologous Recombination
  • Mutation*
  • Pectobacterium carotovorum / genetics
  • Pectobacterium carotovorum / virology
  • Plasmids / genetics