A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

• Published in: Synthetic and systems biotechnology Vol. 9; no. 4; pp. 658 - 666
• Main Authors: Yang, Zhiheng, Li, Bixiao, Bu, Ruihong, Wang, Zhengduo, Xin, Zhenguo, Li, Zilong, Zhang, Lixin, Wang, Weishan
• Format: Journal Article
• Language: English
• Published: China Elsevier B.V 01.12.2024; KeAi Publishing Communications Ltd; KeAi Publishing; KeAi Communications Co., Ltd
• Subjects: Alternative energy; Bacteria; CRISPR; Deletion; Efficiency; Enzymes; Factories; Genetic diversity; Genetic engineering; Genomes; Genomics; gRNA; Long-range genomic deletions; Metabolic engineering; Non-homologous end joining; Original; Parageobacillus thermoglucosidasius; Phylogenetics; Plasmids; Proteins; Thermophile; Thermostable NHEJ enzymes; Type I CRISPR system; Type I CRISPR system; Thermostable NHEJ enzymes; Thermophile; Long-range genomic deletions
• ISSN: 2405-805X; 2405-805X
• DOI: 10.1016/j.synbio.2024.05.009

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool—through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy—we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.