TY - JOUR
T1 - Optimized nickase- and nuclease-based prime editing in human and mouse cells
AU - Adikusuma, Fatwa
AU - Lushington, Caleb
AU - Arudkumar, Jayshen
AU - Godahewa, Gelshan I.
AU - Chey, Yu C. J.
AU - Gierus, Luke
AU - Piltz, Sandra
AU - Geiger, Ashleigh
AU - Jain, Yatish
AU - Reti, Daniel
AU - Wilson, Laurence O. W.
AU - Bauer, Denis C.
AU - Thomas, Paul Q.
N1 - Copyright the Author(s) 2021. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.
PY - 2021/10/11
Y1 - 2021/10/11
N2 - Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one 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.
AB - Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one 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.
UR - http://www.scopus.com/inward/record.url?scp=85118286698&partnerID=8YFLogxK
U2 - 10.1093/nar/gkab792
DO - 10.1093/nar/gkab792
M3 - Article
C2 - 34534334
AN - SCOPUS:85118286698
SN - 0305-1048
VL - 49
SP - 10785
EP - 10795
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 18
ER -