Institute of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
The State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, 200438, China.
Sci Rep. 2017 May 24;7(1):2320. doi: 10.1038/s41598-017-02456-y.
Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.
人类多能干细胞 (hPSCs) 为理解复杂特征和疾病的分子机制提供了独特的机会。CRISPR/Cas9 是一种将基因突变引入 hPSCs 进行功能丧失研究的强大工具。在这里，我们开发了一种基于附加体载体的 CRISPR/Cas9 系统，称为 epiCRISPR，用于 hPSCs 中高效的基因敲除。epiCRISPR 系统可实现高达 100%的插入/缺失 (indel) 率。此外，epiCRISPR 系统还可实现高效的双基因敲除和基因组缺失。为了最大限度地减少脱靶切割，我们将附加体载体技术与双缺口策略和最近开发的高保真 Cas9 相结合。因此，epiCRISPR 系统为 hPSCs 中的遗传分析提供了一个高效的平台。
