Prime Assembly Approach Transforms Gene Therapy With Large DNA Inserts

New technology enables the insertion of a large segment of DNA into a genome, potentially expanding gene therapy treatment from cancellation of disease-causing mutations to replacement of an entire gene, scientists say.

Reporting today (April 29, 2026) in Nature, the researchers describe building upon a technique called prime editing by inserting DNA that attaches to the genome through a series of overlapping flaps. This method, which they call a prime assembly approach, avoids a bottleneck in the gene therapy field – a double-strand break to the donor DNA that can cause toxicity and kill cells.

Using this method, we are doing genome assembly rather than making a small edit in a gene. If we think of the genome as a book, we can remove one paragraph and replace it with a new one – or even rewrite a chapter."

Bin Liu, co-lead author of the study and assistant professor of biological chemistry and pharmacology, The Ohio State University College of Medicine

This distinction is important: Because some diseases involve hundreds of mutations, pursuit of a gene therapy treatment would require hundreds of gene edits that would be subject to individual federal approval, Liu said.

"The biggest impact of this technology is we can correct 1,000 heterogeneous mutations at once," he said.

In the study using mammalian cells, Liu and co-lead authors from the University of Massachusetts Chan Medical School show the technique can allow for efficient insertion of a DNA segment containing up to 11,000 base pairs – compared to a maximum of about 800 base pairs successfully inserted with other methods.

"We tried to hit the upper limit of the technology to see how large we can go," he said. "Using our method, we envision setting up a universal platform and incorporating a healthy copy of a gene directly into a patient, no matter what mutation they have."

Achieving the insertion of a large DNA segment involved a combination of techniques.

Any healthy "donor" DNA used for this type of therapy can be manufactured in a lab. The researchers used a twin prime editing method to generate programmable flaps on the target DNA that introduces the DNA insertion to the genome. The flaps complement the ends of the donor DNA, avoiding a double-strand break. This insertion does induce a single-strand DNA break, which is considered less likely than a double-strand break to be toxic to a cell, Liu said.

Assays evaluating and visualizing the efficiency of the insertion in mammalian cell cultures demonstrated the method's promise, he said.

The editing steps removed the need for reliance on a repair mechanism, called homology directed repair, that has been a key part of gene-editing techniques that involved cuts to DNA. By ruling out that repair step, which occurs in actively dividing cells, the prime assembly technique can incorporate both single- and double-stranded DNA donors and be used as therapy in non-dividing cells such as neurons and heart cells.

"Previous applications that relied on homology-directed repair have worked well in cells, but in animal models, its efficiency is often very low," Liu said. "That's why our method provides a big advantage by harnessing prime editing."

The team calls the technology "prime assembly" in a nod to the common lab technique "Gibson assembly cloning," which joins DNA in a test tube.

There is more work to do, including determining the best delivery vehicle for the donor DNA segment and delivering editor – likely a lipid nanoparticle or adeno-associated virus. Additionally, testing the effectiveness of in vivo editing is planned in Liu's lab and with collaborators in Ohio State's Gene Therapy Institute, including ophthalmologist Tom Mendel.

The work was co-led by Erik Sontheimer, professor, and Wen Xue, associate professor, in the RNA Therapeutics Institute at UMass Chan Medical School. Additional co-authors include Yanjun Zhang of Ohio State; Andrew Petti, Xuntao Zhou, Haoyang Cheng, Jenny Gao, Matthew Yee, Youwei Qiao, Lin Zhou and Scot Wolfe of UMass Chan Medical School; and Tingting Jiang of Icahn School of Medicine at Mount Sinai.

This work was supported by the National Institutes of Health, the Leducq Foundation Translatlantic Network of Excellence Program and the Cystic Fibrosis Foundation.