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

• Published in: iScience 2022-06, Vol.25 (6), p.104438-104438, Article 104438
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: Bioengineering; Biotechnology; Genetic engineering; Synthetic biology
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2022.104438

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 Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment. [Display omitted] •Transcriptional inactivation of Piga renders mESCs full resistance to proaerolysin•PIGA serves as counterselectable marker in endogenous Piga-inactivated mESCs•Piga function is readily restored by Cre-mediated STOP cassette excision•Conditional knockout and restoration strategy is also applicable to Hprt Biotechnology; Genetic engineering; Bioengineering; Synthetic biology