Chen Liangliang, Ye Ying, Dai Hongxia, Zhang Heyao, Zhang Xue, Wu Qiang, Zhu Zhexin, Spalinskas Rapolas, Ren Wenyan, Zhang Wensheng
Cam-Su Genomic Resource Center, Soochow University, Suzhou 215123, China.
The State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau.
Stem Cells Int. 2018 Jun 14;2018:9576959. doi: 10.1155/2018/9576959. eCollection 2018.
Loss-of-function studies are critically important in gene functional analysis of model organisms and cells. However, conditional gene inactivation in diploid cells is difficult to achieve, as it involves laborious vector construction, multifold electroporation, and complicated genotyping. Here, a strategy is presented for generating biallelic conditional gene and DNA regulatory region knockouts in mouse embryonic stem cells by codelivery of CRISPR-Cas9 and short-homology-arm targeting vectors sequentially or simultaneously. Collectively, a simple and rapid method was presented to knock out any DNA element conditionally. This approach will facilitate the functional studies of essential genes and regulatory regions during development.
功能丧失研究在模式生物和细胞的基因功能分析中至关重要。然而，在二倍体细胞中实现条件性基因失活很困难，因为这涉及到繁琐的载体构建、多次电穿孔以及复杂的基因分型。在此，我们提出了一种策略，通过依次或同时共递送CRISPR-Cas9和短同源臂靶向载体，在小鼠胚胎干细胞中产生双等位基因条件性基因和DNA调控区域敲除。总的来说，我们提出了一种简单快速的方法来条件性敲除任何DNA元件。这种方法将有助于在发育过程中对必需基因和调控区域进行功能研究。
