Published on Fri Jul 02 2021

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Adikusuma, F., Lushington, C., Arudkumar, J., Godahewa, G. I., Chey, Y. C. J., Gierus, L., Piltz, S., Reti, D., Wilson, L., Bauer, D., Thomas, P.

Prime editing holds enormous potential for research and clinical applications. Currently, delivery of PE components to mammalian cell lines requires multiple plasmid vectors. To overcome this limitation, we generated all-in-one prime editing (PEA1) constructs.

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Abstract

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. Currently, the delivery of PE components to mammalian cell lines requires multiple plasmid vectors. To overcome this limitation, 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 generated intended edits with efficiencies that were similar, and in some cases exceeded, the PE-nickase system. We also show that the nuclease prime editor can generate intended modifications in mouse fetuses with up to 100% efficiency.