Nature Biotechnology
(2026) Cite this article
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.
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Raw data of high-throughput sequencing have been deposited in the National Center for Biotechnology Information Sequence Read Archive database under accession code PRJNA1473194 (ref. 66). Data needed to evaluate the conclusions in the study are present in the paper and the Supplementary Information. Uncropped blots and gels are included in the supplementary materials, as are sequences for generating donor libraries. Plasmids generated in this study can be provided by the corresponding author L.W. (lead contact) upon request pending scientific review and a completed material transfer agreement.
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