To explore the feasibility of constructing a tobacco mutant library using the CRISPR/Cas9 gene editing technology,we 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,on-target editing rate,and off-target editing of transgenic offspring using the tobacco variety'Honghua Dajinyuan'as experimental material in this study.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 rate of 77%.(2)Among 77 transgenic offspring carrying sgRNA,69 sgRNAs edited the target genes,with an editing rate 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 high co-transformation rate,high editing rate,and low probability of off-target editing.
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