通过CRISPR/Cas9进行同源重组（HDR）是对基因进行编辑的重要手段，在生物医药领域展现出巨大的应用前景。然而目前HDR发生的频率过低，提高其效率是当前基因编辑领域的研究热点之一。本项目拟通过基因密码子扩展技术，对Cas9蛋白实现定点的生物正交修饰，将HDR的供体DNA与切割复合物实现共价交联，通过增加基因切割位点附近的供体DNA的有效浓度提高HDR的发生频率。在此基础上，我们拟开发蛋白定点双修饰技术，对Cas9蛋白进一步实现定点的双化学修饰，用两种不同的生物正交反应，将供体DNA与荧光分子同时精准修饰到Cas9蛋白上，进一步提高HDR效率。最后我们拟将该技术应用于T细胞的精准改造，为CART的免疫疗法提供新技术。本课题的特点是用化学手段（定点修饰的生物正交反应）来解决生物技术的难题（提高基因编辑中同源重组的效率），发挥化学生物学学科特色，最终实现基础科研与应用的有机结合。

Homology-directed repair (HDR) through CRISPR/cas9 is an important gene editing method and showed great therapeutic potential. However, HDR is limited by its low efficiency. To solve this problem, we intended to use an expanded genetic code strategy to site-specifically incorporate bio-orthogonal reactive amino acids into Cas9 proteins to afford a covalent Cas9-donor DNA conjugate. Such conjugation will bring the donor DNA to the cleavage site and effectively increase the local concentration for an enhanced HDR efficiency. In addition, we will explore the possibility of duo functionalities incorporation using an further improved expanded genetic code method, which will allow two mutually orthogonal reactive groups to be modified simultaneously onto Cas9 protein for a more enhanced HDR efficiency. Finally, with such a chemical biology tool, our goal is to apply this new technology to the precise genetic editing of T cells for CART therapeutics.