Protocol for genomic recombineering in Yersinia ruckeri using CRISPR Cas12a coupled with the λ Red system

Genomic manipulation of Yersinia ruckeri, a pathogen of salmonid fish species, is essential for understanding bacterial physiology and virulence. Here, we present a protocol for genomic recombineering in Y. ruckeri, a species reluctant to standard genomic engineering, using CRISPR Cas12a coupled with the λ Red system. We describe steps for identifying protospacer guides, preparing repair template plasmids, and electroporating Yersinia cells with Cpf1 and protospacer plasmids with homologous arms. We then detail procedures for genome editing and plasmid curing. [Display omitted] •A robust, user-friendly protocol for generating genetic mutants in Yersinia ruckeri•Generation of precise gene edits employing CRISPR Cas12a coupled with the λ Red system•Detailed description of plasmid cloning, customizable for varying gene editing needs•Protocol optimized for bacterial species reluctant to standard genome editing techniques Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Genomic manipulation of Yersinia ruckeri, a pathogen of salmonid fish species, is essential for understanding bacterial physiology and virulence. Here, we present a protocol for genomic recombineering in Y. ruckeri, a species reluctant to standard genomic engineering, using CRISPR Cas12a coupled with the λ Red system. We describe steps for identifying protospacer guides, preparing repair template plasmids, and electroporating Yersinia cells with Cpf1 and protospacer plasmids with homologous arms. We then detail procedures for genome editing and plasmid curing.