Bacteriophage genome engineering with CRISPR-Cas13a

Jumbo phages such as Pseudomonas aeruginosa.KZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous `phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the.KZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.

Automated summary

This study provides a new genetic engineering tool for DNA jumbo phages by combining homologous recombination with RNA-targeting CRISPR-Cas13a enzyme. The results showed successful insertion, deletion, and fluorescent tagging of genes in the KZ genome. Additionally, important findings such as the fluorescent tagging of the protein gp93 during phage DNA ejection and the impact of deleting a specific protein on phage burst size were discovered. This study holds significance in addressing the challenge of genetic editing in phages.