Construction of a mutant library associated with aroma genes in tobacco using CRISPR/Cas9

Subjects: Biology >> Botany >> Applied botany submitted time 2023-12-24 Cooperative journals: 《广西植物》

ZENG Wanli LIANG Gang GAO Qian LI Yuanchuan XU li JIANG Jiarui XU Yong XIANG Haiying

Abstract： To explore the feasibility of constructing a tobacco mutant library using the CRISPR/Cas9 gene editing technology, the study designed sgRNAs of 100 aroma related genes in tobacco, constructed a plasmid library composed of 100 corresponding CRISPR/Cas9 editing vectors, and analyzed the co-transformation rate, target editing efficiency, and off-target editing rate of transgenic offspring. The results were as follows: (1) After co-transformation of 100 sgRNAs mediated by Agrobacterium tumefaciens, 77 of them were detected in 172 positive transformation strains, with a co-transformation efficiency of 77%; (2) Among 77 transgenic offspring carrying sgRNA, 69 sgRNAs edited the target genes, with an editing efficiency of 89.6%; (3) Sequencing detection revealed that only one sgRNA produced off-target editing at a non-target site, indicating a very low probability of off-target editing of CRISPR/Cas9 in tobacco. In summary, it is feasible to construct a mutant library by the co-transformation of a CRISPR/Cas9 vector library to edit a large number of candidate target genes in tobacco. This method has the characteristics of the high co-transformation efficiency, the high editing efficiency, and the low probability of off target editing.

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