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

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

    Research output: Contribution to journalArticlepeer-review

    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 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.

    Original languageEnglish (US)
    Article number104438
    JournaliScience
    Volume25
    Issue number6
    DOIs
    StatePublished - Jun 17 2022

    Keywords

    • Bioengineering
    • Biotechnology
    • Genetic engineering
    • Synthetic biology

    ASJC Scopus subject areas

    • General

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