Design and artificial synthesis of gRNA library for genetic breeding of Saccharomyces cerevisiae
- 1Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding, College of Biology and Food Engineering, Anhui Polytechnic University, Wuhu, 241000, China
Abstract
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.
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BibTeX
@article{wang2026design,
title = {{Design and artificial synthesis of gRNA library for genetic breeding of Saccharomyces cerevisiae}},
author = {Xinyi Wang and Peiyan Ma and Majda Hanioui and Zhangnian Wang and Qianqian Yu and Abdul Waheed and Kun Liu and Yan Liu and Zhenglian Xue and Ming Zhao},
journal = {Biodesign research},
year = {2026},
month = sep,
doi = {10.1016/j.bidere.2026.100123},
url = {https://europepmc.org/article/PMC/PMC13717876},
note = {CC BY},
}
RIS
TY - JOUR
TI - Design and artificial synthesis of gRNA library for genetic breeding of Saccharomyces cerevisiae
AU - Xinyi Wang
AU - Peiyan Ma
AU - Majda Hanioui
AU - Zhangnian Wang
AU - Qianqian Yu
AU - Abdul Waheed
AU - Kun Liu
AU - Yan Liu
AU - Zhenglian Xue
AU - Ming Zhao
PY - 2026
DA - 2026/09/28
JO - Biodesign research
DO - 10.1016/j.bidere.2026.100123
UR - https://europepmc.org/article/PMC/PMC13717876
AB - 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.
SN - 2693-1257
ER -