Transposase-Assisted Donor Tethering Boosts Large-Fragment HDR in Plants

Precise insertion of large DNA fragments by homology-directed repair (HDR) remains inefficient and poorly reproducible in plants, largely due to limited donor availability at double-strand break sites. Here, we develop a transposase-assisted donor tethering strategy that improves the reliability of HDR-mediated large-fragment insertion. By fusing Cas9 to an integration-defective piggyBac variant that retains sequence-specific DNA-binding activity, donor templates are physically co-localized with Cas9-induced breaks. When combined with a transcription-coupled donor and a repair-pathway-biased Cas9 variant, this system enhances the frequency of accurate large-fragment insertions. Using this approach, we achieved efficient and precise kilobase-scale targeted gene insertions across multiple loci in both dicot and monocot species. These findings establish donor tethering as an effective strategy to improve plant HDR efficiency and provide a general framework for precise large-fragment genome insertion.