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快速但不急躁:一种用于基因组编辑细胞的简化选择方法。

Fast but not furious: a streamlined selection method for genome-edited cells.

机构信息

IUF-Leibniz Research Institute for Environmental Medicine, Core Unit Model Development, Düsseldorf, Germany.

Genome Engineering and Measurement Laboratory, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

出版信息

Life Sci Alliance. 2021 Apr 26;4(6). doi: 10.26508/lsa.202101051. Print 2021 Jun.

Abstract

In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods.

摘要

在过去的十年中,转录激活因子样效应物核酸酶和基于 CRISPR 的基因组工程已经彻底改变了我们的生物学研究方法。由于其高效性和易用性,几乎每个实验室现在都可以轻松地开发定制的敲除和敲入动物或细胞模型。然而,基因修饰细胞的产生通常需要一个选择步骤,通常通过抗生素或荧光标记来实现。选择筛选标记取决于可用的实验室资源,例如细胞类型,并且还应考虑时间和成本等参数。在这里,我们提出了一种称为磁激活基因组编辑细胞分选的新的快速策略,该策略基于 Cas9 阳性细胞表面抗原(即 tCD19)的磁性分选能力来选择基因修饰细胞。通过使用磁激活基因组编辑细胞分选,我们成功地生成并分离了基因修饰的人诱导多能干细胞、原代人成纤维细胞、SH-SY5Y 神经母细胞瘤样细胞、HaCaT 和 HEK 293T 细胞。我们的策略通过提供一种快速、廉价且易于使用的替代方法,扩展了基因组编辑工具包。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4721/8127327/55d44f7ceb26/LSA-2021-01051_FigS1.jpg

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