Donor-complementary prime editing enables precise kilobase and library-compatible DNA insertions.

Yunzheng Fang, Jingyao Tang, Jiawei Xi, Binfeng Yang, Feng Zhang, Lingbo Wang

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Article in Nature biotechnology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

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5 · Who and what money

Authors and funding

6 authors.

Yunzheng Fang *Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China.

Jingyao Tang *Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China.ORCID http://orcid.org/0000-0002-8950-3709

Jiawei XiShanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China.

Binfeng YangShanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China.

Feng ZhangChildren's Hospital of Fudan University, National Children's Medical Center, Institute of Medical Genetics and Genomics, Human Phenome Institute, Fudan University, Shanghai, China. zhangfeng@fudan.edu.cn.ORCID http://orcid.org/0000-0003-4556-8276

Lingbo WangShanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Obstetrics and Gynecology Hospital, State Key Laboratory of Genetics and Development of Complex Phenotypes, Fudan University, Shanghai, China. wanglingbo@fudan.edu.cn.ORCID http://orcid.org/0000-0002-0962-8457

Funding

No grant is acknowledged in the PubMed record.

6 · The paper itself

Abstract

Methods for precise genomic DNA insertion that avoid double-strand breaks (DSBs) are constrained by limited throughput or the need for multistep editing. Here we report donor-complementary prime editing (DoPE), which combines a 3'-overhang double-stranded DNA (odsDNA) donor with a pair of overhang-complementary prime editing guide RNAs (opegRNAs) and a PE2* prime editor to achieve precise insertion of DNA sequences up to 12.5 kilobases (kb). Using one opegRNA pair and donor pools constructed from synthesized single-stranded oligonucleotides, we demonstrate in situ saturation mutagenesis across a targeted EGFP region at both amino acid and nucleotide resolutions. DoPE employing short (approximately 30-nucleotide) overhangs supports various insertions ranging from small fragments to those exceeding 10 kb. Furthermore, we replace mutant exons of PRKCSH, either individually or simultaneously, to correct diverse mutations, establishing a mutation-agnostic approach that corrects distinct alleles uniformly in vitro. Our study demonstrates DoPE as a one-step, DSB-free and library-compatible method for precise insertion of large DNA fragments without requiring recombinases or transposases.

Identifiers

PMID42665684

DOI10.1038/s41587-019-0032-3

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