Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR.

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

STAR protocols 2022 Jun 17

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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. © 2022 The Author(s).

Citation

Yuhan Liu, Qian Chen, Chenglin Song, Zhen Xu, Shuting Yang, Xiajun Li. Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR. STAR protocols. 2022 Jun 17;3(2):101436