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

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Bacteriophage engineering is crucial for applications but selecting specific genotypes after minor modifications is difficult.
• Existing methods face challenges with subtle sequence distinctions and protospacer-adjacent motif limitations.

Purpose of the Study:

• To develop a two-phase CRISPR-Cas-based phage engineering approach for precise genetic modifications in Pectobacterium carotovorum phage ZF40.
• To enable selection and quantification of engineered phage variants in mixed populations.

Main Methods:

• Utilized homologous recombination via engineered plasmids containing homologous sequences and a mini-CRISPR array.
• Employed an endogenous type I-E CRISPR-Cas system for counter-selection against non-recombinant phage genomes.
• Incorporated distinct 20-bp barcodes as differential target sites for programmed CRISPR-Cas activity.

Main Results:

• Successfully deleted target genes and restored modified loci in P. carotovorum phage ZF40.
• Overcame challenges related to sequence distinction and protospacer-adjacent motif requirements for CRISPR-Cas counter-selection.
• Enabled accurate quantification of engineered phage variants within mixed populations using barcodes.

Conclusions:

• The developed two-phase CRISPR-Cas engineering method facilitates precise and subtle genetic modifications in bacteriophages.
• This approach is valuable for studies and applications requiring mixtures of genetically similar phages.
• The method enhances the utility of bacteriophages by enabling sophisticated genetic engineering and variant tracking.