Design and artificial synthesis of gRNA library for genetic breeding of Saccharomyces cerevisiae

CRISPR mediated genome engineering strategies were developed and applied for genome-wide mutagenesis. However, high-throughput gRNA libraries require design and synthesis, which limits their economy and versatility. In this study, we designed in vitro transcription strategy for artificially synthesizing random gRNA libraries, which were transformed into cells to guide the diverse mutations generated through Cas protein induced double stranded DNA breaks. As a proof of concept, the Saccharomyces cerevisiae strain C0 with overexpression of tHMG1 and β-carotene biosynthesis key genes was used as the original strain, and the β-carotene synthesis ability of strain C0 was increased to that of 5.8 times after three rounds of iterative mutation. Furthermore, for the mutated S . cerevisiae strain C7-143 with high-level β-carotene production, a mutant strain AgC8-174 with a 26% increase in β-carotene yield was obtained through one round of mutation. This strategy completely avoided the limitations of plasmid library, significantly shortened the experimental cycle, and improved the versatility of gRNA library across different species. The results fully demonstrated the feasibility of the technology and lays a solid foundation for its application in the field of microbial genetics and breeding.