CRISPR/CAS9诱导猪基因组损伤修复机制研究及其在精准基因编辑中的应用

CRISPR/CAS9技术通过靶向诱导基因组断裂损伤，启动自身损伤修复通路，使基因编辑猪的效率大幅提高。然而由于介导精准编辑的同源重组修复发生概率远远低于随机剪切插入的非同源末端连接修复，导致CRISPR/CAS9介导基因定点插入、突变等精准编辑的效率仍然较低。大量研究表明，通过调控宿主细胞基因组损伤修复通路，增强其同源重组修复水平，可有效提高精准基因编辑的效率；且不同的物种间DNA损伤修复机制存在一定差异。因此本研究拟应用单细胞和转录组测序技术，揭示猪体细胞及胚胎在Cas9损伤后的特异性修复机制，筛选其高效特异性通路调控元件；并以特异性关键元件为靶点调控修复通路，增强同源重组修复，从而提高猪体细胞及胚胎细胞的精准编辑效率，为精准基因编辑猪的高效制备奠定基础。

With the introduction of genome editing using CRISPR/CAS9 technology, the efficiency of porcine genome editing has been increased dramatically owing to inducing DNA double strand breaks (DSBs) and stimulating repair pathway. However, the efficiency of precise genome editing ( such as knockin, point mutation) was relative low because the DSBs were repaired mainly by error-prone nonhomologous end joining pathway, but not the precise repair pathway-homologous recombination(HR). Many research indicated that the efficiency of precision genome editing could be improved by modulating DNA repair pathways and increasing HR activity. And the DNA repair mehanism differed among different species. Therefore here we intend to study the specific DSBs repair mechanism of pig and find out the key and high efficient factors that control the repair pathway, then improve the porcine precise genome editing with those key targets or factors. The research will help to improve porcine precise genome editing greatly.