Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR

Yuhan Liu; Qian Chen; Chenglin Song; Zhen Xu; Shuting Yang; Xiajun Li

doi:10.1016/j.xpro.2022.101436

Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR

STAR Protoc. 2022 Jun 7;3(2):101436. doi: 10.1016/j.xpro.2022.101436. eCollection 2022 Jun 17.

Authors

Yuhan Liu^{1

2}, Qian Chen¹, Chenglin Song¹, Zhen Xu¹, Shuting Yang¹, Xiajun Li^{1

3}

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China.
³ Genome Editing Center, ShanghaiTech University, Shanghai 201210, China.

Abstract

Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.

Keywords: CRISPR; Genetics; Molecular Biology; Stem Cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems* / genetics
Embryonic Stem Cells
Mice
Mouse Embryonic Stem Cells*
Puromycin / pharmacology
Transfection

Substances

Puromycin