gRNA二级结构对拟南芥HD2基因CRISPR/Cas9编辑率的影响

Effect of Secondary Structure of gRNA Adaptor on Editing Efficiency of CRISPR/Cas9——A Case from The HD2 Gene of Arabidopsis thaliana

近年来,利用基因编辑工具编辑特定基因逐渐成为一种获得突变体的主要方法。CRISPR/Cas9编辑技术是目前最新和最高效的基因编辑技术。然而,这一技术在植物中的编辑率受到很多因素的影响,例如如何设计gRNA以及如何避免脱靶效应等。为了探究gRNA的二级结构对拟南芥基因编辑的影响,我们选择了拟南芥基因组中的组蛋白去乙酰化酶(HDACs)基因家族中一个植物特有的亚家族(HD2)为靶基因,通过设计这一亚家族中3个HD2基因不同的CRISPR/Cas9靶点,观察不同gRNA接头二级结构对CIRSPR/Cas9编辑率的影响。结果显示:不同gRNA接头二级结构对基因的编辑率会有一定的影响,当二级结构由一段单链RNA、5个茎环、2个突环和3个发夹环组成的时候,编辑率最高。该研究可为后续人们使用以tRNA为策略设计gRNA的CRISPR/Cas9工具时选择合适的靶点,为提高基因编辑率提供基础。

The acquisition of mutants is one of the most important prerequisites for the study of gene function. In recent years, using gene editing tools to edit specific genes has gradually become a major method to obtain mutants. CRISPR/Cas9 is the latest and most efficient gene editing technology. However, the editing efficiency of this technology in plants is affected by many factors. For example, how to design gRNA and how to avoid off-target effect. In order to explore the effect of secondary structure of gRNA on gene editing in Arabidopsis thaliana, we selected a plant specific subfamily(HD2) in the HDACs gene family of Arabidopsis genome as the target gene. By designing different CRISPR/Cas9 targets of three HD2 genes in this subfamily, we observed the effects of different secondary structures of gRNA adaptors on the efficiency of success CRISP/Cas9 editing. The results showed that different secondary structure of gRNA adaptor had a certain effect on the efficiency of gene editing. When the secondary structure consists of a single strand RNA, five stem loops, two bulge loops and 3 hairpin loops, the editing efficiency is the highest. This study will help us to choose the appropriate target when we design CRISPR/Cas9 tool based on tRNA strategy, so as to improve the success efficiency of gene editing.