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
Affiliations
Weimin Zhang
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Ran Brosh
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Laura H. McCulloch
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Yinan Zhu
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Hannah Ashe
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Gwen Ellis
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Brendan R. Camellato
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA
Sang Yong Kim
Department of Pathology, NYU Langone Health, New York, NY 10016, USA
Matthew T. Maurano
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA; Department of Pathology, NYU Langone Health, New York, NY 10016, USA
Jef D. Boeke
Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA; Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA; Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, USA; Corresponding author
Summary: 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.
