Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia.

Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of species. This study demonstrates the development and application of a genome-engineering tool in two strains, and , which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.