Abstract

We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et?al.¹.