光敏感gRNA介导的光敏感CRISPR/Cas9系统的构建及其对靶基因编辑的精准动态光调控

CRISPR介导的基因编辑已经被广泛用于生命科学和医学的研究。在基因敲除、定点突变等领域得到广泛的应用，在构建疾病模型和纠正基因缺陷上极具临床应用价值。但其本身也同样存在编辑效率低和脱靶效应的问题。本项目拟对CRISPR系统中的关键gRNA进行精准的修饰，通过在5'端引入光敏感基团和靶向基团，构建靶向基团修饰的光敏感gRNA，利用靶向基团与其受体蛋白质结合产生的大位阻效应，抑制gRNA与Cas9和靶DNA形成CRISPR复合物。在特定的时间和空间范围内，通过光诱导激活gRNA及其介导的CRISPR系统，实现在时间和空间上对基因编辑的精准和动态光调控，降低可能的基因编辑脱靶效应。

CRISPR induced gene editing has been widely used in life science and medical research, esp in the fields of gene knockdown, specific gene mutation et al. These functions demonstrated that CRISPR can be used to construct disease models and correct mutated genes, which shows very promising apllications in clinics. However, CRISPR itself also shows the low gene editing yield as well as off-target effect. This proposal plans to precise modification of gRNA which is the key component of CRISPR gene editing system. By Inducing a photolabile linker and targeting moiety to gRNA, a photolabile gRNA was rationally designed to temporally mask gRNA activity and its following CRISPR complex formation with Cas9 and target DNA. At the specific time and location, gRNA and its CRISPR system can be activated through simple light activaiton. The goal of this proposal is to develop a new gRNA tool and realize the precise and dynamic photoregulation of target gene editing in spatiotemporal resolution, which will possibly reduce the off-target effect of gene editing.