可调控CRISPR-Cas系统发展及用于核酸表观遗传修饰动态调控研究

Nucleic acids are important macromolecules in the continuity of life. Importantly, nucleic acids carry a variety of different chemical modifications, which have profound effects on their properties. Moreover, epigenetic modifications can significantly alter chromatin structure and nucleic acid accessibility. The epigenetic modifications of nucleic acids are thought to be highly dynamic and play important roles in dynamic regulation of gene expression. Therefore, further progress in the use of nucleic acid epigenetic modifications as targets to regulate gene expression requires the use of a dynamically controllable regulatory system. At present, the CRISPR/Cas systems represent a commonly used tool for gene manipulation. Because of their high efficiency and system diversity, they have been widely used for gene regulation at both DNA and RNA levels. However, the time and spatial specificity of CRISPR/Cas systems needs to be improved. In previous studies, most researchers have focused on the engineering of Cas proteins. The challenges include technical difficulties and limitations in real-time regulation. This project aims at the development of chemical regulatory CRISPR/Cas systems based on the engineering of gRNA. The efficient post-synthesis modification of gRNA will allow for the development of chemical regulatory CRISPR/Cas systems, which can meet the requirements for real-time and precise regulation of gene expression at multi-dimensional levels. Moreover, this project will use the controllable CRISPR/Cas system to study the function of m6A in life cycle of viruses.

核酸是重要的生物大分子，发生在核酸上的化学修饰存在深远影响，DNA和RNA上的表观遗传修饰就是重要的一类。核酸表观修饰在生命过程中动态变化、动态平衡，并以动态调控基因表达，因此以核酸表观修饰为靶标的基因调控，乃至于疾病相关性研究，需要动态可控的调控工具系统。目前，CRISPR/Cas系统是一类常用的基因操纵工具，其高效性和体系的多元性，对于DNA、RNA层面的基因调控靶标具有应用价值。但时空特异性和可控性问题需要提高，这也是此前研究者持续关注的问题。但以往研究集中在对Cas蛋白的改造，具有技术难度和实时调控上的局限性。本项目拟基于CRISPR/Cas系统的gRNA进行化学调控体系的开发，gRNA的易修饰易改造性，有利于发展多元化的调控体系，以满足基因层面、转录及转录后等多层面实时精确调控的需求。在此基础上，本项目将利用所发展的可控CRISPR/Cas系统，开展病毒相关m6A机制研究。

项目主要研究内容是建立核酸动态化学修饰调控的，可诱导基因编辑体系及相关应用研究，重点围绕建立可诱导CRISPR/Cas系统，通过“可控诱导激活”的策略，来减少普通CRISPR/Cas系统由于持续活性状态，而产生的对非靶标的非特异作用，即脱靶效应。按照计划任务书，构建了系列基于RNA修饰-去修饰化学调控的可诱导CRISPR系统，并有效用于细胞内的可诱导基因编辑过程。通过较为系统的探究，建立了有效的RNA小分子靶向调控体系，可望后续拓展至动物层面基因编辑调控研究和基于RNA化学靶向的生物医学应用。