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

Autor:	Zhang W; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Brosh R; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., McCulloch LH; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Zhu Y; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Ashe H; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Ellis G; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Camellato BR; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA., Kim SY; Department of Pathology, NYU Langone Health, New York, NY 10016, USA., Maurano MT; Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA.; Department of Pathology, NYU Langone Health, New York, NY 10016, USA., Boeke JD; 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.
Jazyk:	angličtina
Zdroj:	IScience [iScience] 2022 May 23; Vol. 25 (6), pp. 104438. Date of Electronic Publication: 2022 May 23 (Print Publication: 2022).
DOI:	10.1016/j.isci.2022.104438
Abstrakt:	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. Competing Interests: Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to Re-Open Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. (© 2022 The Authors.)
Databáze:	MEDLINE
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