Prime assembly with linear DNA donors enables large genomic insertions

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.