Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

文献类型： 外文期刊

第一作者： Pan, Wenbo

作者： Pan, Wenbo;Zhang, Huawei;Pan, Wenbo;Liu, Xue;Li, Dayong;Liu, Xue;Li, Dayong

作者机构：

关键词： genome editing; CRISPR; Cas9; intron-mediated enhancement; GRF5; lettuce

期刊名称：FRONTIERS IN PLANT SCIENCE （ 影响因子：6.627； 五年影响因子：7.255 ）

ISSN： 1664-462X

年卷期： 2022 年 13 卷

页码：

收录情况： SCI

摘要： The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of Cas9 are commonly used to improve the editing efficiency. However, these strategies also increase the risk of off-target mutation. Here, we developed a new strategy to utilize intron-mediated enhancement (IME)-assisted 35S promoter to drive Cas9 and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both Cas9 and sgRNA. In addition, we developed another strategy to enrich cells highly expressing Cas9/sgRNA by co-expressing the developmental regulator gene GRF5, which has been proved to ameliorate the transformation efficiency, and the transgenic plants from these cells also exhibited enhanced editing efficiency. This system elevated the genome editing efficiency from 14-28% to 54-81% on three targets tested in lettuce (Lactuca sativa) without increasing the off-target editing efficiency. Thus, we established a new genome editing system with highly improved on-target editing efficiency and without obvious increasement in off-target effects, which can be used to characterize genes of interest in lettuce and other crops.

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