Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates

Bin Liu; Xiaolong Dong; Chunwei Zheng; David Keener; Zexiang Chen; Haoyang Cheng; Jonathan K Watts; Wen Xue; Erik J Sontheimer

doi:10.1038/s41587-023-01947-w

Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates

Nat Biotechnol. 2023 Sep 14. doi: 10.1038/s41587-023-01947-w. Online ahead of print.

Authors

Bin Liu^#¹, Xiaolong Dong^#^{1

2}, Chunwei Zheng^#¹, David Keener¹, Zexiang Chen¹, Haoyang Cheng¹, Jonathan K Watts^{1

3

4}, Wen Xue^{5

6

7

8}, Erik J Sontheimer^{9

10

11

12}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
² Tessera Therapeutics, Somerville, MA, USA.
³ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁴ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁵ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁶ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁷ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁸ Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
¹⁰ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
¹¹ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
¹² Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.

^# Contributed equally.

PMID: 37709915
DOI: 10.1038/s41587-023-01947-w

Abstract

Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).