真核生物基因组高度折叠形成有序并层次分明的拓扑结构域，以适应有限体积的细胞核。染色质架构蛋白CTCF和cohesin通过介导不同调控元件之间的染色质环化，精确调控基因的时空特异性表达，但CTCF/cohesin建立的染色质高级结构在维持基因组完整性方面的影响还不清楚。申请人最近开发了CRISPR 遗传学DNA片段编辑方法，发现单个CTCF位点的相对方向会影响染色质拓扑结构环的方向，并提出了CTCF/cohesin介导的染色质高级结构的分子组装模型。本项目通过利用CRISPR片段编辑技术从基因组和蛋白组层面调控CTCF/cohesin介导染色质高级结构，以架构染色体高级结构的cohesin“环挤出”调控DNA修复核心的建立为模型假设，阐明染色质高级结构影响基因组精准修复的分子机制，为最终实现精准的基因组DNA片段编辑奠定基础。

The eukaryotic genome is highly folded into 3D genome architectures, forming an ordered and hierarchical topologically associated domains within the limited cell nucleus. The architectural proteins CTCF and cohesin, involving in the formation of chromatin looping between distinct regulatory elements, precisely regulate the spatiotemporal gene expression. However, whether CTCF/cohesin-mediated chromatin topological architectures have an impact on maintaining genomic integrity remains obscure. We recently developed a CRISPR DNA-fragment editing method and found single CTCF sites determine specific long-distance chromatin looping between distal enhancers and target promoters, proposing an asymmetric blocking model of cohesin sliding by directional CTCF binding. In this grant application, using CRISPR DNA-fragment editing method to manipulate CTCF/cohesin-mediated chromatin architectures from the genome and proteome level, we could elucidate the mechanism of precise DNA repair regulated by 3D genome architectures and investigate the relationship between the efficiency of precise DNA-fragment editing and the 3D genome architectures. In particular, based on the hypothesis that organization of 3D genome architectures through cohesin-dependent “loop extrusion” determine the formation of DNA repair foci, we could achieve precise CRISPR DNA-fragment editing which has important implications regarding the maintenance of genome integrity through chromatin architectural proteins CTCF or cohesin.