1. Academic Validation
  2. Prime assembly with linear DNA donors enables large genomic insertions

Prime assembly with linear DNA donors enables large genomic insertions

  • bioRxiv. 2025 Jun 17:2025.06.16.659978. doi: 10.1101/2025.06.16.659978.
Bin Liu 1 Andrew Petti 1 Xuntao Zhou 1 Haoyang Cheng 1 Lin Zhou 2 Tingting Jiang 3 Erik J Sontheimer 1 4 5 6 Wen Xue 1 2 4 5
Affiliations

Affiliations

  • 1 RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
  • 2 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
  • 3 Icahn Genomics Institute, Department of Immunology and Immunotherapy, Department of Oncological Sciences, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
  • 4 Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA.
  • 5 Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA.
  • 6 Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Abstract

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime editing (PE) guide RNAs (pegRNAs) have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a Prime Assembly (PA) approach for the insertion of large DNA donor fragments, whose ends are designed to overlap with the flaps generated by twinPE. We used PA to insert one, two, or three overlapping DNA fragments, with total insertion sizes ranging from 0.1 to 11 kilobase pairs. An inhibitor of non-homologous end joining (NHEJ) enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced and does not require co-delivery of exogenous DNA-dependent DNA polymerases. Our study demonstrates that PA can initiate "Gibson-like" assembly in cells to generate gene insertions without double-stranded DNA breaks or recombinases.

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