用于先进基因组编辑应用的小鼠胚胎干细胞中的条件性反选择敲除。

A conditional counterselectable knockout in mouse embryonic stem cells for advanced genome writing applications.

作者信息

Zhang Weimin, Brosh Ran, McCulloch Laura H, Zhu Yinan, Ashe Hannah, Ellis Gwen, Camellato Brendan R, Kim Sang Yong, Maurano Matthew T, Boeke Jef D

机构信息

Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA.

Department of Pathology, NYU Langone Health, New York, NY 10016, USA.

出版信息

iScience. 2022 May 23;25(6):104438. doi: 10.1016/j.isci.2022.104438. eCollection 2022 Jun 17.

DOI:10.1016/j.isci.2022.104438

PMID:35692632

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9184564/

Abstract

Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional knockout cells show similar proaerolysin resistance as full (non-conditional) deletion cells, which enables the use of a transgene as a counterselectable marker for genome engineering purposes. Native function is readily restored in conditional knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous . Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.

摘要

覆盖可反向选择标记是去除野生型DNA或将其替换为感兴趣的有效载荷DNA的有效策略。目前，哺乳动物高效基因组工程的一个瓶颈是缺乏能够在不影响生物体发育潜能的情况下稳健发挥作用的可反向选择（负选择）标记。在此，我们报告了一种条件性敲除策略，该策略能够在小鼠胚胎干细胞中实现基于气单胞菌溶素原的高效反向选择。条件性敲除细胞表现出与完全（非条件性）缺失细胞相似的气单胞菌溶素原抗性，这使得能够将转基因用作基因组工程目的的可反向选择标记。在条件性敲除细胞中，天然功能很容易恢复，以促进后续的小鼠发育。我们还通过对内源基因进行条件性敲除证明了我们策略的通用性。综上所述，我们的工作为先进的小鼠基因组编辑和小鼠模型建立提供了一种新工具。