Pan Wenbo, Liu Xue, Li Dayong, Zhang Huawei
Peking University Institute of Advanced Agricultural Science, Weifang, China.
School of Advanced Agricultural Sciences, Peking University, Beijing, China.
Front Plant Sci. 2022 Jun 22;13:930592. doi: 10.3389/fpls.2022.930592. eCollection 2022.
The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of 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 promoter to drive and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both and sgRNA. In addition, we developed another strategy to enrich cells highly expressing /sgRNA by co-expressing the developmental regulator gene , 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 () 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.
CRISPR/Cas9基因组编辑系统在许多作物中的效率仍然有限。利用强启动子来提高基因的表达水平是常用的提高编辑效率的方法。然而，这些策略也增加了脱靶突变的风险。在这里，我们开发了一种新策略，利用内含子介导增强（IME）辅助启动子在单个转录本中驱动基因和sgRNA，通过适度增强基因和sgRNA的表达来提高编辑效率。此外，我们还开发了另一种策略，通过共表达发育调控基因来富集高表达基因/sgRNA的细胞，该基因已被证明可改善转化效率，来自这些细胞的转基因植物也表现出更高的编辑效率。该系统在生菜（）中测试的三个靶点上，将基因组编辑效率从14%-28%提高到了54%-81%，而没有增加脱靶编辑效率。因此，我们建立了一种新的基因组编辑系统，其靶向编辑效率得到了高度提高，且脱靶效应没有明显增加，可用于鉴定生菜和其他作物中感兴趣的基因。
