Prime assembly with linear DNA donors enables large genomic insertions

Area of Science:

• Molecular Biology
• Genome Engineering
• Gene Therapy

Background:

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

Purpose of the Study:

• To develop a novel approach for efficient insertion of large DNA fragments using prime editing.
• To overcome the limitations of existing methods for large DNA insertions.

Main Methods:

• Developed a prime assembly (PA) approach utilizing overlapping DNA fragments designed to anneal with twin prime editing (twinPE) generated flaps.
• Administered an inhibitor of non-homologous end joining to enhance insertion efficiency and precision.
• Demonstrated PA's ability to insert single or multiple overlapping DNA fragments ranging from 0.1 kb to 11 kb.

Main Results:

• Successfully inserted large DNA fragments (0.1–11 kb) using the PA approach.
• Observed enhanced efficiency and precision of insertions when using a non-homologous end joining inhibitor.
• Showcased PA's independence from canonical homology-directed repair pathways and its ability to proceed in non-cycling cells.

Conclusions:

• Prime assembly (PA) is an effective method for inserting large DNA fragments, ranging from 0.1 kb to 11 kb.
• PA facilitates Gibson-like assembly within cells, enabling gene insertions without requiring double-stranded DNA breaks, recombinases, or homology-directed repair.
• The developed method offers a promising advancement for genome engineering and gene therapy applications.