crRNA-Cas12a复合体识别与切割靶DNA分子机制的单分子生物物理学研究

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. One Cas protein, Cas9, has been shown to be a new tool in genome-editing applications. To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA (CRISPR RNA) and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The crRNA are small RNAs transcribed from CRISPR loci, and used to guide effector endonucleases that target invading DNA based on sequence complementarity. However, Cas9 has also been found to have certain defects and limitations, such as off-target effects. In 2015, Zhang Feng's research team discovered a new Cas protein - Cas12a (also known as cpf1). Cas12a is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Different from cas9, Cas12a is guided by a single guide RNA (gRNA), and has high activity and low off-target effect. It is considered to be a new generation of CRISPR gene editing tools. The Cas12a-RNA complex recognizes and forms a base-complementary pairing structure with the target DNA, the process of which must accompanied by the adjustment of its own conformation. This project intends to study the kinetics of recognition and cleavage of target DNA by crRNA-Cas12a complex using conventional biochemistry, single-molecule fluorescence, single-molecule manipulation and computational biology. We expect to discover the key amino acids or nucleobases, secondary structures, protein domains and their dynamic properties during DNA digestion. In this way, we can understand what processes these parts have participated in and what role they have played. We can summarize the conformational-function model of the complex recognition and cutting target DNA. The understanding of the mechanisms of each part can help us to provide structural basis for the design of new CRISPR/Cas system with low off-target effect.

CRISPR-Cas系统是细菌及古细菌为抵御病毒入侵而产生的后天免疫机制。该系统中的效应蛋白Cas9已成为当前基因编辑的有力工具，但也暴露出一定的缺陷和局限性，如严重的脱靶效应。张峰课题组在2015年发现了Cas12a蛋白（又称cpf1），其具有活性高、脱靶效应低的特点，被认为是新一代CRISPR基因编辑工具。crRNA-Cas12a复合物要识别并与靶DNA形成碱基互补配对结构，其过程必然伴随着自身构象的调整。本项目拟利用常规生物化学、单分子荧光、单分子操纵技术以及计算生物学等多种手段，研究crRNA-Cas12a复合体识别并切割靶DNA的动力学过程，以期发现关键氨基酸或碱基、二级结构、蛋白结构域在该过程中的动态特性、参与到的具体过程以及对整体复合物功能的影响，并在深入了解各部分运行机制的情况下总结该复合物识别切割靶DNA的构象变化模型，为进一步优化该基因编辑工具提供理论基础。

CRISPR-Cas系统是细菌及古细菌为抵御病毒入侵而产生的后天免疫机制。该系统中的效应蛋白Cas9已成为当前基因编辑的有力工具，但也暴露出一定的缺陷和局限性，如严重的脱靶效应。近期发现了Cas12a蛋白（又称cpf1），其具有活性高、脱靶效应低的特点，被认为是新一代CRISPR基因编辑工具。crRNA-Cas12a复合物要识别并与靶DNA形成碱基互补配对结构，其过程必然伴随着自身构象的调整。我们利用常规生物化学、单分子荧光、单分子操纵技术以及计算生物学等多种手段，研究crRNA-Cas12a复合体识别并切割靶DNA的动力学过程，并在深入了解各部分运行机制的情况下总结该复合物识别切割靶DNA的构象变化模型。我们的主要研究进展包括：1）研究总结出了Cas12a/crRNA/DNA三元复合物R-Loop的形成过程及对靶DNA的切割机制；2）通过研究发现了潜在的抑制CRISPR-Cas12a系统的蛋白；3）与合作者合作，完成基于 CRISPR/Cas9 的文库筛选系统筛选并发现能改善HCC患者临床反应的与合成致死性相关的基因。我们的研究为进一步优化该基因编辑工具提供理论基础。