Protocol for genomic recombineering in Yersinia ruckeri using CRISPR Cas12a coupled with the 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 lambda 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.