New gene editing method enables full gene replacement in one step

08:00

By: Dakir Madiha

New gene editing method enables full gene replacement in one step

Scientists have developed a gene editing technique capable of inserting DNA segments more than 13 times larger than previous limits, opening a path toward replacing entire defective genes in a single therapeutic intervention. The method, known as prime assembly, was published on April 29, 2026 in Nature and marks a significant advance in genome engineering.

The approach was designed by a team led by Bin Liu at the Ohio State University College of Medicine, in collaboration with researchers from UMass Chan Medical School. It builds on twin prime editing, a CRISPR-based system that generates programmable single-stranded DNA flaps at a targeted genomic site. A linear DNA donor with overlapping ends is then integrated into the genome through a process similar to Gibson assembly, carried out directly inside living cells.

Using this technique, researchers achieved insertion efficiencies of up to 50 percent for a donor sequence of 0.8 kilobases. They also successfully inserted DNA fragments as large as 11.3 kilobase pairs into human cells, including the full gene associated with Duchenne muscular dystrophy. Previous prime editing approaches struggled to exceed insertions of 400 to 800 base pairs, limiting their clinical potential.

A key advantage of prime assembly is that it avoids double-strand DNA breaks, which are commonly used in conventional CRISPR-based gene insertion methods. These breaks can trigger cell death and activate tumor suppressor pathways, restricting their use in therapeutic settings. By relying only on single-strand cuts, the new method reduces cellular toxicity and does not depend on homology-directed repair, a mechanism active mainly in dividing cells. This expands its potential use to non-dividing cells such as neurons and cardiomyocytes, which have historically been difficult targets for gene therapy.

The team also demonstrated that adding a DNA-PK inhibitor, AZD-7648, improved both the efficiency and precision of DNA insertion. Unintended genetic modifications were reduced to levels close to background noise, addressing a major challenge in gene editing safety.

Rather than correcting individual mutations one by one, prime assembly could allow clinicians to replace an entire disease-causing gene with a healthy version in a single step. This strategy could address a wide range of genetic disorders with diverse mutation profiles. As proof of concept, researchers inserted a CAR gene into the T cell receptor locus, a step relevant to cancer immunotherapy.

The next phase of research will focus on testing safety and efficacy in animal models. Delivering the gene editing components inside the body remains a central challenge, with lipid nanoparticles and adeno-associated viruses under investigation as potential delivery systems. If validated, the method could reshape the scope of gene therapy by enabling large-scale, precise genome modifications in a broader range of cell types.