Wu Yun, Xu Kun, Ren Chonghua, Li Xinyi, Lv Huijiao, Han Furong, Wei Zehui, Wang Xin, Zhang Zhiying
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
Department of Biology, Zun Yi Normal College, Zunyi, Guizhou, China.
FEBS Lett. 2017 Mar;591(6):903-913. doi: 10.1002/1873-3468.12599. Epub 2017 Mar 8.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome-modified cells is often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporter cassettes into paired donor plasmids, which were used as both the surrogate reporters and the knock-in donors. By applying our dual surrogate reporter-integrated donor system, we demonstrate high frequency of CRISPR/Cas9-mediated biallelic genome integration in both human HEK293T and porcine PK15 cells (34.09% and 18.18%, respectively). Our work provides a powerful genetic tool for assisting the selection and enrichment of cells with targeted biallelic genome modification.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）系统最近已成为一种简单却强大的基因组工程工具，已被广泛用于各种生物体和细胞类型的基因组修饰。然而，筛选双等位基因基因组修饰细胞通常既耗时又具有技术挑战性。在本研究中，我们将两个不同的替代报告盒整合到配对的供体质粒中，这些质粒既用作替代报告基因，又用作敲入供体。通过应用我们的双替代报告基因整合供体系统，我们证明了在人HEK293T细胞和猪PK15细胞中CRISPR/Cas9介导的双等位基因基因组整合频率很高（分别为34.09%和18.18%）。我们的工作为辅助选择和富集具有靶向双等位基因基因组修饰的细胞提供了一种强大的遗传工具。
