An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.
An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.
复制标题
基于游离载体的 CRISPR/Cas9 系统，可在人类多能干细胞中高效进行基因敲除
DOI：
10.1038/s41598-017-02456-y
复制
发表时间：
2017-05-24
影响因子：
4.6
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Xie Y;Wang D;Lan F;Wei G;Ni T;Chai R;Liu D;Hu S;Li M;Li D;Wang H;Wang Y
人类多能干细胞（hPSC）代表了理解复杂性状和疾病的分子机制的独特机会。CRISPR/Cas9是将基因突变引入hPSC中进行功能丧失研究的有力工具。在这里，我们开发了一种基于附加型载体的CRISPR/Cas9系统，我们称之为epiCRISPR，用于hPSC中的高效基因敲除。epiCRISPR系统能够产生高达100%的插入/缺失（indel）率。此外，epiCRISPR系统能够实现有效的双基因敲除和基因组缺失。为了最小化脱靶切割，我们将附加型载体技术与双切口策略和最近开发的高保真Cas9相结合。因此，epiCRISPR系统为hPSC中的遗传分析提供了高效的平台。
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.