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Updated: Sep 15, 2025

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Prime assembly with linear DNA donors enables large genomic insertions.

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Summary

Prime Assembly (PA) enables large DNA insertions for genome engineering, overcoming limitations of twin prime editing (PE). This method efficiently inserts large DNA fragments using overlapping templates and an NHEJ inhibitor, avoiding double-stranded DNA breaks.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Targeted insertion of large DNA fragments is crucial for genome engineering and gene therapy.
  • Existing prime editing (PE) methods struggle with efficient insertion of DNA fragments larger than 400 base pairs.

Purpose of the Study:

  • To develop a novel method, Prime Assembly (PA), for efficient insertion of large DNA fragments.
  • To enhance the efficiency and precision of large DNA insertions using PA.

Main Methods:

  • Developed Prime Assembly (PA) utilizing overlapping DNA donor fragments designed to ligate with twin prime editing (PE)-generated flaps.
  • Administered an inhibitor of non-homologous end joining (NHEJ) to improve insertion outcomes.
  • Inserted single, double, or triple overlapping DNA fragments ranging from 0.1 to 11 kilobase pairs.

Main Results:

  • Successfully inserted large DNA fragments (up to 11 kilobase pairs) using the PA approach.
  • Demonstrated enhanced insertion efficiency and precision with the addition of an NHEJ inhibitor.
  • Showcased PA's ability to perform "Gibson-like" assembly within cells, generating gene insertions without double-stranded DNA breaks or recombinases.

Conclusions:

  • Prime Assembly (PA) is an effective method for inserting large DNA fragments, significantly improving upon existing twin prime editing (PE) capabilities.
  • PA offers a versatile and efficient platform for gene insertion applications in genome engineering and potentially gene therapy.
  • The method's reliance on easily produced DNA templates and avoidance of double-stranded DNA breaks or exogenous polymerases makes it a promising tool.