Abstract：
Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.
摘要：
表观遗传编辑,也称为EpiEdit，提供了一种令人兴奋的方法来控制基因表达而不改变DNA序列。在这项研究中,我们评估了EpiEdit在植物启动子中的应用，特别是MLO(霉菌基因座o)基因启动子。我们使用改进的CRISPR-(D)Cas9系统，其中核酸酶缺陷型Cas9（dCas9）与表观遗传修饰剂融合，实验证明该工具用于在体内植物表观基因组编辑之前优化植物启动子的表观基因工程的实用性。引导RNA用于将dCas9-表观遗传修饰融合蛋白递送至靶基因序列，它诱导MLO基因表达的修饰。我们使用克隆到荧光素酶报告系统中的植物启动子进行初步实验，将其转染到人系统中并使用双荧光素酶报告基因测定进行分析。结果表明，这种方法可能在植物表观基因组编辑的早期阶段有用。因为它可以帮助在植物系统条件下进行体内实验之前选择对植物启动子的适当修饰。总的来说,结果证明了基于CRISPR(d)Cas9的EpiEdit对基因表达的精确和受控调控的潜力。
