CRISPR基因编辑技术的DNA双链断裂修复机制研究

The CRISPR (clustered regularly-interspaced short palindromic repeats) genome editing technology is one of the most important scientific breakthroughs in recent years. Its development has transformed the field of biology and has shown great potential and possible economic benefits in applications in biology, agriculture and medicine. As CRISPR executes genome editing through various DNA double strand break (DSB) repair mechanisms, one strategy of improving its efficiency and precision is to control the choice of the DSB repair pathways. Our current understanding of DSB repair mechanisms is largely based on studies of DSB repair with 3'-protruding ends. However, known CRISPR nucleases generate DSBs with blunt ends (e.g. Cas9) and 5’-protruding ends (e.g. Cpf1), which were difficult to generate in a site-specific manner in mammalian cells in order to study repair of such ends in the past. As a result, although our knowledge in DSB repair has been greatly improving, our understanding of unique DSB repair of CRISPR remains limited. Nevertheless, our studies have indicated: 1) Mammalian cells may differently regulate DSB repair for 5’-protruding ends, blunt ends and 3’-protruding ends; 2) ATM, DNA-PKcs and ATR, three central phosphatidylinositol 3-kinase-related kinases (PIKKs) in DNA damage response, appear to play an important role in this regulation; 3) Histone H2AX, a common substrate for these three PIKKs and a key factor initiating the chromatin response to DSBs, also assists some of these repair mechanisms. Based on these studies, we thus hypothesize that different DNA end structures (5’-protruding ends, blunt ends and 3’-protruding ends) in DSBs may activate different PIKKs to establish H2AX-dependent chromatin response with different composition, and to determine the selection of DSB repair pathways and regulation of the selected pathway. To test this hypothesis, we propose to study the role of ATM, DNA-PKcs, ATR and H2AX in the DSB repair pathways homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA), upon DSBs induced by I-SceI (generating 3’-protruding ends) and by CRISPR nucleases Cas9 and Cpf1. The objective of this proposal is to not only clarify the unique characteristics of DSB repair regulation for CRISPR at the molecular level, but also reveal the mechanisms of DSB repair with different DNA end structures. This will not only better and deepen our understanding of DSB repair in general and unique DSB repair for CRISPR in particular, but also provide new opportunities for the improvement of the CRISPR technology.

新兴的CRISPR基因编辑技术已显示巨大的应用潜力和可能的经济效益。通过控制其DNA双链断裂（DSB）修复原理是目前改良该技术的新方向之一。前期工作显示，CRISPR核酸酶制造的DNA末端是该技术DSB修复机制独特性的一个决定性因素。结合先前对DSB损伤应答相关的三个中心激酶ATM、DNA-PKcs和ATR以及组蛋白H2AX功能的认识，我们推测，不同的DNA末端结构（3’外伸端、平末端或5’外伸端）可能激活不同的中心激酶，建立不同组成的H2AX依赖的染色质应答区，最终决定DSB修复途径的选择与调节。因此，通过研究细胞内ATM、DNA-PKcs、ATR和H2AX在CRISPR基因编辑中对四条不同DSB修复途径的调节，特别是通过比较分析3’外伸端、平末端和5’外伸端的DSB修复分子机制，阐明CRISPR DSB修复机制的独特性。这不仅将推动DSB修复机制的全面了解，也将为改良该技术提供新机会。

CRISPR基因编辑技术主要依赖于CRISPR/Cas9-sgRNA复合物能够在细胞内定点诱导DNA双链断裂（DSB）的特性，利用细胞本身的DSB修复途径产生所需要的编辑产物。然而，CRISPR核酸酶诱导DSB的过程及产生的DSB末端具有其独特性，DSB修复机制也应该独具特色。利用我们建立的各类同源重组（HDR）和非同源末端连接（NHEJ）报告系统，结合遗传学、细胞生物学、生物化学及生物信息学等研究手段，通过研究ATM、DNA-PKcs、ATR和γH2AX-MDC1信号轴在Cas9诱导的DSB修复中的作用及机制，我们发现：.1）γH2AX-MDC1信号轴在Cas9切割产生的DSB末端协助断裂末端附近的染色质重塑，推动经典型NHEJ（c-NHEJ）修复途径的参与，但产生额外的碱基丢失；2）像I-SceI诱导产生的3’-粘性末端，CRISPR/Cas9诱导的平末端也偏向精准型NHEJ修复，于是在此基础上，设计开发了一个可以提高编辑效率和精准度的基于配对Cas9-sgRNA的CRISPR基因编辑技术；3）ATM在I-SceI、Cas9和Cpf1诱导的HDR中作用不同，这个不同也会在小鼠细胞和人源细胞之间改变；4）CRISPR靶点滞留调控DSB修复途径选择，导致不同靶点的修复途径选择不同，产生的基因编辑突变异质性也随之改变；5）c-NHEJ抑制剂或c-NHEJ因子缺失将导致CRISPR基因编辑脱靶效应提高，为此，我们开发了基于dCas9的定点抑制NHEJ的方法，提高基因敲进效率和避免脱靶效应。.该项目不仅揭示了CRISPR/Cas9诱导的DSB修复的独特性调节，还在此基础上建立了新的基因编辑策略，可以提高基因编辑效率和精准度，避免脱靶效应。.