Cas9
化学
连接器
基因组编辑
清脆的
融合蛋白
引导RNA
基因组工程
细胞生物学
DNA
核酸内切酶
计算生物学
基因组
生物化学
基因
生物
重组DNA
操作系统
计算机科学
作者
Jun Yin,Shan Hou,Qun Wang,Lichen Bao,Dingkang Liu,Yali Yue,Wenbing Yao,Xiangdong Gao
标识
DOI:10.1021/acs.bioconjchem.9b00022
摘要
Successful and efficient delivery of Cas9 protein and gRNA into cells is critical for genome editing and its therapeutic application. In this study, we developed an improved supercharged polypeptide (SCP) mediated delivery system based on dithiocyclopeptide linker to realize the effective genome editing in tumor cells. The fusion protein Cas9-linker-SCP (Cas9-LS) forms positively charged complexes with gRNA in vitro to provide possibilities for gRNA delivery into cells. Under the microenvironment of tumor cells, the dithiocyclopeptide linker, containing matrix metalloproteinase 2 (MMP-2) sensitive sequence and an intramolecular disulfide bond, can be completely disconnected to promote the release of Cas9 protein with the nuclear localization sequence (NLS) in the cytoplasm and transfer to the cell nucleus for highly efficient genome editing, resulting in an obvious increase of indel efficiency in comparison to fusion protein without dithiocyclopeptide linker (Cas9-SCP). Furthermore, Cas9-LS shows no significant cytotoxicity and minimal hemolytic activity. We envision that the microenvironment-responsive Cas9 protein delivery system can facilitate more efficient genome editing in tumor cells.
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