Nature biotechnologyAug 28, 2026

Donor-guided prime editing allows precise insertion of long and multiple DNA sequences

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Aug 30, 2026

Abstract

Precise insertion of DNA sequences up to 12.5 kilobases is achieved using donor-complementary prime editing (DoPE).

DoPE combines a double-stranded DNA donor with overhang-complementary guide RNAs and a prime editor.
This method allows for various insertions, including small fragments and those exceeding 10 kb.
In situ saturation mutagenesis was demonstrated across a targeted region with both amino acid and nucleotide resolution.
DoPE can replace mutant exons of the PRKCSH gene, correcting various mutations uniformly in vitro.
The approach is characterized as one-step, avoids double-strand breaks, and is compatible with libraries.

Simplified

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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Donor-guided prime editing allows precise insertion of long and multiple DNA sequences

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