基于CRISPR-Cas9技术研究PUMA调控同源重组修复蛋白RAD51表达的作用机制

CRISPR-Cas9 system for the gene modification such as hematopoietic stem cells or somatic cells holds a great promise in the gene therapy of blood disease. However, how to quickly repair DNA damage and enhance the efficiency of homologous recombination repair is the key to its successful applications in the clinic. Our previous studies demonstrated that Puma-deficiency led to a better survival rate associated surprisingly with reduced DNA damage and apoptosis in iPSC. In addition, Puma-deficient stem and progenitor cells showed higher DNA double strand breaks repair activity and expression of Rad51 after radiomimetic DNA damage. It suggested that PUMA may play an important role in the system of CRISPR-Cas9. Thus, in our current proposal, the system of CRISPR-Cas9 is used as model. The PUMA and the key protein of RAD51 in homologous recombination repair are chosen as the important factors of mechanism. We plan to firstly identify the dynamic change of PUMA in the process of DNA double-strand break repair. Then, the potential molecular mechanisms will be explored by using the technology of RNA-Seq. Our results will improve the efficiency of gene editing and provide a theoretical basis for the gene therapy of blood diseases.

用CRISPR-Cas9技术改造基因突变的造血干细胞或体细胞在血液系统疾病的治疗中有巨大潜力，如何快速修复DNA损伤、提高打靶效率是其临床应用需要突破的关键技术瓶颈。我们的前期工作表明，下调Puma可以降低诱导多能干细胞重编程过程中DNA的损伤和细胞凋亡；小鼠胚胎干细胞和造血祖细胞经放射造成DNA损伤后，抑制Puma可以显著提高DNA双链断裂修复的比例和Rad51蛋白的表达水平。这提示我们PUMA可能在CRISPR-Cas9系统基因修饰的过程中发挥了重要作用。本研究以CRISPR-Cas9系统为研究模型，以PUMA和同源重组修复中关键蛋白RAD51作为切入点，探究PUMA在DNA双链断裂修复过程中的动态变化和作用，同时利用RNA-Seq技术阐明PUMA在DNA损伤修复中潜在的分子机制，为进一步提高打靶效率、探索血液系统疾病基因治疗新手段提供理论依据。

用CRISPR-Cas9技术改造基因突变的造血干细胞或体细胞在血液系统疾病的治疗中有巨大潜力，如何快速修复DNA损伤、提高打靶效率是其临床应用需要突破的关键技术瓶颈。我们的前期工作表明，下调Puma可以降低诱导多能干细胞重编程过程中DNA的损伤和细胞凋亡；小鼠胚胎干细胞和造血祖细胞经放射造成DNA损伤后，抑制Puma可以显著提高DNA双链断裂修复的比例和Rad51蛋白的表达水平。这提示我们PUMA可能在CRISPR-Cas9系统基因修饰的过程中发挥了重要作用。本项目明确了PUMA在DNA双链断裂修复过程中发挥的作用，我们发现PUMA 敲降后，可以显著提高CRISPR-Cas9系统下293T和iPS的编辑效率，即可以提高CRISPR-Cas9 系统中同源重组修复的比例，这一研究结果可以为血液系统疾病的基因治疗提供新手段，最终为提高造血干细胞打靶效率提供实验基础。