Optimized nickase- and nuclease-based prime editing in human and mouse cells
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(Published version)
Date
2021
Authors
Adikusuma, F.
Lushington, C.
Arudkumar, J.
Godahewa, G.I.
Chey, Y.C.J.
Gierus, L.
Piltz, S.
Geiger, A.
Jain, Y.
Reti, D.
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Journal article
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Nucleic Acids Research (NAR), 2021; 49(18):10785-10795
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Fatwa Adikusuma, Caleb Lushington, Jayshen Arudkumar, Gelshan I. Godahewa, Yu C.J. Chey, Luke Gierus, Sandra Piltz, Ashleigh Geiger, Yatish Jain, Daniel Reti, Laurence O.W. Wilson, Denis C. Bauer, and Paul Q. Thomas
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Abstract
Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-inone prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.
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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.