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

doi:10.1016/j.isci.2022.104438

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

Biomedical Engineering

Research output: Contribution to journal › Article › peer-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 language	English (US)
Article number	104438
Journal	iScience
Volume	25
Issue number	6
DOIs	https://doi.org/10.1016/j.isci.2022.104438
State	Published - Jun 17 2022

Keywords

Bioengineering
Biotechnology
Genetic engineering
Synthetic biology

ASJC Scopus subject areas

General

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10.1016/j.isci.2022.104438

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title = "A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications",

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.",

keywords = "Bioengineering, Biotechnology, Genetic engineering, Synthetic biology",

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

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doi = "10.1016/j.isci.2022.104438",

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AU - Zhang, Weimin

AU - Brosh, Ran

AU - McCulloch, Laura H.

AU - Zhu, Yinan

AU - Ashe, Hannah

AU - Ellis, Gwen

AU - Camellato, Brendan R.

AU - Kim, Sang Yong

AU - Maurano, Matthew T.

AU - Boeke, Jef D.

PY - 2022/6/17

Y1 - 2022/6/17

N2 - 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.

AB - 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.

KW - Bioengineering

KW - Biotechnology

KW - Genetic engineering

KW - Synthetic biology

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

Abstract

Keywords

ASJC Scopus subject areas

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