Protocol for genomic recombineering in Yersinia ruckeri using CRISPR Cas12a coupled with the λ Red system
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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.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 103014 |
| Tidsskrift | STAR Protocols |
| Vol/bind | 5 |
| Udgave nummer | 2 |
| Antal sider | 17 |
| ISSN | 2666-1667 |
| DOI | |
| Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:
The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (NNF14CC0001). N.M.I.T. acknowledges support from a DFF Research Project 1 grant (1030-00289B) and an NNF Hallas-M\u00F8ller Emerging Investigator grant (NNF17OC0031006). N.M.I.T. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. E.M.S.-R. acknowledges the support of Lundbeckfonden through a postdoctoral fellowship (R346-2020-1683). The pAC-crRNA plasmid was a kind gift from Yi-Cheng Sun, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. The schematic in Figure 1A was partly created using vectors from BioRender. R.N.E. designed and carried out all experiments, with support from E.M.S.-R. and input from N.M.I.T. E.M.S.-R. supported R.N.E. in idea, project planning, and supervision with input from N.M.I.T. The authors declare no competing interests.
Funding Information:
The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation ( NNF14CC0001 ). N.M.I.T. acknowledges support from a DFF Research Project 1 grant ( 1030-00289B ) and an NNF Hallas-M\u00F8ller Emerging Investigator grant ( NNF17OC0031006 ). N.M.I.T. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. E.M.S.-R. acknowledges the support of Lundbeckfonden through a postdoctoral fellowship ( R346-2020-1683 ). The pAC-crRNA plasmid was a kind gift from Yi-Cheng Sun, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. The schematic in Figure 1 A was partly created using vectors from BioRender.
Publisher Copyright:
© 2024 The Authors
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