Abstract
Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report several nCas9-DNA polymerase fusion proteins and their engineered versions to create a simple and efficient two-component chimeric oligonucleotide-directed editing (CODE) system. CODE contains a derivative of Bst DNA polymerase engineered for increased thermostability and processivity as well as a chimeric pegRNA (cpegRNA) for programmable search and replace genome editing. Additionally, CODEMax(exo+) features a 5’ to 3’ exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate that CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci in HEK293T cells with plasmid- and RNP-based delivery. We also show that CODEMax can successfully modify mouse and bovine embryos with up to 9.3% precise editing. Further optimization of CODEMax systems may enhance editing outcomes in embryos and other challenging contexts. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.
Similar content being viewed by others
Data availability
All data supporting the study are available within the main text, supplementary information, and supplementary data files. Source data are also included in this paper. Sequencing data have been deposited to NCBI database with the accession number PRJNA1266727. Source data are provided with this paper.
References
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023).
Gould, S. I. Prime editing sensors enable multiplexed genome editing. Nat. Rev. Genet. https://doi.org/10.1038/s41576-024-00737-7 (2024).
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
Vats, S., Kumar, J., Sonah, H., Zhang, F. & Deshmukh, R. Prime editing in plants: prospects and challenges. J. Exp. Bot. https://doi.org/10.1093/jxb/erae053 (2024).
Zhao, Z., Shang, P., Mohanraju, P. & Geijsen, N. Prime editing: advances and therapeutic applications. Trends Biotechnol. 41, 1000–1012 (2023).
Zeng, H. et al. Recent advances in prime editing technologies and their promises for therapeutic applications. Curr. Opin. Biotechnol. 86, 103071 (2024).
Ponnienselvan, K. et al. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic Acids Res. 51, 6966–6980 (2023).
Zhang, W. et al. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. Elife 12, RP90948 (2024).
Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).
Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002 e3926 (2023).
Antoniou, P. et al. Modified pegRNAs mitigate scaffold-derived prime editing by-products. Nat. Commun. 16, 3374 (2025).
Lesnik, E. A. & Freier, S. M. Relative thermodynamic stability of DNA, RNA, and DNA: RNA hybrid duplexes: relationship with base composition and structure. Biochemistry 34, 10807–10815 (1995).
Liu, B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01947-w (2023).
da Silva, J. F. et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Nat. Biotechnol. 43, 923–935 (2025).
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).
Abdus Sattar, A. K., Lin, T. C., Jones, C. & Konigsberg, W. H. Functional consequences and exonuclease kinetic parameters of point mutations in bacteriophage T4 DNA polymerase. Biochemistry 35, 16621–16629 (1996).
Reha-Krantz, L. J. & Nonay, R. L. Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases. J. Biol. Chem. 269, 5635–5643 (1994).
Qi, R. & Otting, G. Mutant T4 DNA polymerase for easy cloning and mutagenesis. PLoS ONE 14, e0211065 (2019).
Jordan, C. S. & Morrical, S. W. Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA-binding protein. DNA Repair (Amst.) 25, 41–53 (2015).
Pant, K. et al. The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: kinetic, single-molecule, and cross-linking studies. PLoS ONE 13, e0194357 (2018).
Wang, Y. et al. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res. 32, 1197–1207 (2004).
Shehi, E. et al. The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity. FEBS Lett. 497, 131–136 (2001).
Reha-Krantz, L. J., Woodgate, S. & Goodman, M. F. Engineering processive DNA polymerases with maximum benefit at minimum cost. Front Microbiol. 5, 380 (2014).
Li, V., Hogg, M. & Reha-Krantz, L. J. Identification of a new motif in family B DNA polymerases by mutational analyses of the bacteriophage t4 DNA polymerase. J. Mol. Biol. 400, 295–308 (2010).
Leavitt, M. C. & Ito, J. T5 DNA polymerase: structural-functional relationships to other DNA polymerases. Proc. Natl. Acad. Sci. USA. 86, 4465–4469 (1989).
Frey, M. W., Nossal, N. G., Capson, T. L. & Benkovic, S. J. Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3′->5′ exonuclease activity. Proc. Natl. Acad. Sci. USA 90, 2579–2583 (1993).
Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63 (2000).
Oscorbin, I. & Filipenko, M. Bst polymerase - a humble relative of Taq polymerase. Comput. Struct. Biotechnol. J. 21, 4519–4535 (2023).
Paik, I., Bhadra, S. & Ellington, A. D. Charge engineering improves the performance of Bst DNA polymerase fusions. ACS Synth. Biol. 11, 1488–1496 (2022).
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 e5629 (2021).
Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018).
Liang, Z., Wu, Y., Guo, Y. & Wei, S. Addition of the T5 exonuclease increases the prime editing efficiency in plants. J. Genet. Genom. 50, 582–588 (2023).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Wang, Y. et al. N(6)-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation. Mol. Cell 83, 3818–3834 e3817 (2023).
Iyyappan, R. et al. Single-nucleotide resolution epitranscriptomic profiling uncovers dynamic m(6)A regulation in bovine preimplantation development. bioRxiv https://doi.org/10.1101/2025.07.07.663558 (2025).
Kim-Yip, R. P. et al. Efficient prime editing in two-cell mouse embryos using PEmbryo. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-02106-x (2024).
Yu, G. et al. Prediction of efficiencies for diverse prime editing systems in multiple cell types. Cell 186, 2256–2272 e2223 (2023).
Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).
Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023).
Gu, B., Posfai, E. & Rossant, J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat. Biotechnol. 36, 632–637 (2018).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Georgiadis, M. M. et al. Mechanistic implications from the structure of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase. Structure 3, 879–892 (1995).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Wang, J., Yu, P., Lin, T. C., Konigsberg, W. H. & Steitz, T. A. Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. Biochemistry 35, 8110–8119 (1996).
Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Unciuleac, M. C., Goldgur, Y. & Shuman, S. Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD(+)-dependent polynucleotide ligases. Proc. Natl. Acad. Sci. USA. 114, 2592–2597 (2017).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature https://doi.org/10.1038/s41586-024-07487-w (2024).
Acknowledgements
We would like to thank members of Jain, Myhrvold, Toettcher, Jiang, Adamson, and Burdine labs for insightful discussion and valuable suggestions. We are grateful for the Department of Molecular Biology, Omenn-Darling Bioengineering Institute, the Genomic Core at Princeton University, the NextGen DNA Sequencing core facility at the University of Florida (UF) Interdisciplinary Center for Biotechnology Research (ICBR), and UF Health Cancer Center and the UF Herbert Wertheim College of Engineering for their support. This work was financially supported in part by the National Institutes of Health (NIH), Grant T32GM136583 (N.R.), the NIH-NIGMS Maximizing Investigator’s Research Award (MIRA) R35GM147788 (P.K.J.), the NIH-NIAID R61AI181016 (P.K.J.), Dinesh O. Shah endowed professorship (P.K.J.), Exxon Mobil Gator Alumni Faculty endowed professorship (P.K.J.), NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development R01HD102533 (Z.J.) and R01HD113698 (Z.J.), NIH award number RM1HG009490 (B.A.), CHDI Foundation (B.A.), the China Scholarship Council (CSC) based on the April 2015 Memorandum of Understanding between the CSC and Princeton University (J.Y.), NIH award number U01DK127429 (J.E.T.) and R01GM144362 (J.E.T.), the New Jersey Commission on Cancer Research (NJCCR) #COCR24PRG007 (R.D.B.), NIH award number T32GM148739 (C.B.Y.), the Princeton Omenn-Darling Bioengineering Institute - Innovators (PBI2) program (L.N.), the Centers for Disease Control and Prevention award 75D30122C15113 (C.M.), and Princeton University (B.A., R.D.B., C.B.Y., C.M., L.N., J.Y., Y.J. and J.E.T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies including the NIH or the CDC. Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.
Author information
Authors and Affiliations
Contributions
L.N., N.R., C.M. and P.K.J. conceptualized the ideas. L.N., N.R., C.M., and P.K.J. designed research. L.N., N.R., B.P., R.I., C.Y., Y.H., K.T.B., A.F., S.G.A., K.V.A., C.M.D.P. and J.G.L. carried out experiments. R.I. and Z.J. carried out the gene editing experiments in the mouse and bovine embryos. C.M., P.K.J., J.E.T., Z.J., B.A., R.D.B., J.Y. and Y.J. helped troubleshoot the experiments and provided suggestions. L.N., N.R., B.P. and R.I. wrote the manuscript, with input from all co-authors. The manuscript was approved by all authors.
Corresponding authors
Ethics declarations
Competing interests
L.N., N.R., B.P., C.M. and P.K.J. have filed patent applications with the USPTO/PCT (WIPO) related to chimeric oligonucleotide-directed genome editing and Cas-polymerase-based programmable genome editing technologies described in this work (PCT/US/2025/036584- pending, published; 63/668,340- converted; PCT/US2024/044167- pending, published; 63/600,216- converted; 63/579,160- converted). P.K.J. is a co-founder of CasNx, LLC and CRISPR, LLC. C.M. is a co-founder of Carver Biosciences. B.A. is/was an advisory board member with financial interest in Arbor Biotechnologies and Tessera Therapeutics. J.Y., Y.J., and B.A. are listed as inventors on patent application(s) related to prime editing technologies. J.E.T. is a scientific advisor for Prolific Machines and Nereid Therapeutics. The remaining authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Nguyen, L.T., Rakestraw, N.R., Pizzano, B.L.M. et al. Efficient genome editing with chimeric oligonucleotide-directed editing. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71624-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-71624-4
