| Apr 30, 2026 |
A new prime assembly technique inserts DNA segments up to 11,000 base pairs into the genome, enabling correction of thousands of mutations at once.
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(Nanowerk News) 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.
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Reporting in Nature (“Prime assembly with linear DNA donors enables large genomic insertions”), 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.
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“Using this method, we are doing genome assembly rather than making a small edit in a gene,” said Bin Liu, a co-lead author of the study and assistant professor of biological chemistry and pharmacology at The Ohio State University College of Medicine. “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.”
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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.
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“The biggest impact of this technology is we can correct 1,000 heterogeneous mutations at once,” he said.
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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.
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“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.”
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Achieving the insertion of a large DNA segment involved a combination of techniques.
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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.
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Assays evaluating and visualizing the efficiency of the insertion in mammalian cell cultures demonstrated the method’s promise, he said.
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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.
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“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.”
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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.
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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.
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