随着ZFN、TALEN及CRISPR/Cas9等基因编辑技术的不断成熟，应用核酸酶对遗传疾病进行的基因治疗逐渐成为人们关注的焦点。在传统的基因治疗中，编码核酸酶的基因通过脂质体转染、核转染及病毒侵染等方式被运输到细胞中去。这些细胞运输方式往往伴随着细胞毒性高、细胞类型依赖性强、外源基因易整合及“脱靶”效应高等缺点。而基于纯化核酸酶蛋白质的基因编辑技术，则可以极大程度上克服上述这些缺点。之前研究中，我们应用纯化的ZFN、TALEN及Cas9蛋白质对包括原代细胞及干细胞在内的多种类型细胞成功进行了基因编辑。此项目中，我们将进一步优化基于核酸酶蛋白质的“绿色”基因编辑技术，提升此技术的效率、降低细胞毒性并探索对原代细胞及干细胞的放大量处理条件，为今后临床应用奠定基础。

The recent advances in ZFN, TALEN and CRISPR/Cas9-baesd gene editing technologies shed the light on the use of nucleases to treat genetic diseases. Traditionally, nuclease-encoding genes are delivered into cells by lipofection, nucleofection or viral transduction. These methods are typically associated with high cytotoxicity, strong cell type dependency, high risk of chromosomal integration and notable off-target effects. Nuclease protein-based gene editing can nevertheless largely eliminate all the above disadvantages. In previous studies, we have successfully employed purified ZFN, TALEN and Cas9 proteins to induce genomic modifications in a wide range of cells including primary and stem cells. In this project, we intend to further optimize this gene-free gene editing technologies. Specifically, we plan to further increase the gene-editing efficiency, decrease cytotoxicity and investigate the clinical-scale treatment of primary and stem cells, aiming to pave the way for the therapeutic application of nuclease proteins.