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Genomic editing holds great potential to treat or cure disease, but the technology continues to have its limitations. For instance, most current methods rely on either untargeted gene delivery or short DNA edits that need to be individualized to each patient.
A team led by Harvard Medical School researchers at Boston Children’s Hospital and Dana-Farber Cancer Institute has now developed a novel genome engineering method called prime assembly that overcomes those two obstacles, allowing long DNA fragments to be stitched into precise and programmable target positions within living cells.
The approach, described Sept. 16 in Nature, may allow for the development of universal gene therapies that can apply to many patients.
Prime assembly builds on the techniques of prime editing, an advanced technology pioneered by Harvard chemist David R. Liu that allows for precise DNA insertions, deletions, and base swaps. Unlike techniques such as CRISPR-Cas9, prime editing makes cuts in just one strand, rather than both strands, of the DNA double helix. This reduces so-called off-target effects, improving safety.
To date, prime editing has been limited to making small- to medium-sized insertions. Many genetic diseases, however, involve hundreds of different mutations, meaning current tools must make many different edits. Prime assembly, by contrast, is designed to correct almost any mutation in a given gene with a single approach.
Prime assembly’s single-step process creates flaps in DNA at specific locations in the genome. The flaps grab hold of new, precisely assembled DNA inserts, which can be one or more gene-sized pieces. These become integrated into the genome as large, permanent edits.
“By using prime editing to write in one flap per strand of the genome, the method controls exactly where the DNA replacement starts and ends,” said co-senior author Daniel Bauer, the HMS Donald S. Fredrickson, MD Associate Professor of Pediatrics at Boston Children’s, director of the Gene Therapy Program at Boston Children’s, and attending physician at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center.
“Because the method is based on prime editing, it is much less likely to cause off-target effects compared to other gene editing methods,” added Bauer, who is also a member of the Harvard Stem Cell Institute and the Broad Institute of MIT and Harvard.
One such off-target effect is the potential for toxicity. Untargeted insertion methods can turn on the wrong genes in the wrong context, raising the risk of cancerous outcomes. Prime assembly’s targeted insertion approach circumvents the risk. Prime assembly also does not rely on DNA double-strand breaks or DNA double-strand donors, both of which can be toxic and cause unwanted cell stress. And while other gene editing methods are mostly limited to dividing cells — which limits therapeutic applications and raises susceptibility to unwanted DNA changes — prime assembly works in nondividing cells.
Building on this milestone, the team is investigating molecular mechanisms that could allow them to engineer even more efficient and precise systems.
As they fine-tune their approach, they hope prime assembly will one day improve patient care.
“We’re working to improve the delivery of the prime assembly components to disease-relevant human cells in vivo, such as hematopoietic stem cells for blood disorder therapies,” said Bauer, whose previous work contributed to the scientific foundations of what became the world’s first approved CRISPR-based gene therapy, for sickle cell disease and beta thalassemia.
The team is also exploring applications of prime assembly to deliver genetic payloads regardless of a patient’s specific mutation to correct “devastating inherited human diseases with unmet clinical needs,” he said.
Adapted from a Boston Children’s news release.
Authorship, funding, disclosures
Sébastien Levesque, Nozomu Kawashima, and Gue-Ho Hwang are co-first authors of the study. Additional authors are Jing Zeng, Vasil Toskov, Timothy Barry, William Mannherz, Luke Homfeldt, Basheer Becerra, Vivien A.C. Schoonenberg, Luca Pinello, and Suneet Agarwal.
The study was supported by the Doris Duke Foundation (#2022092), the St. Jude Children’s Research Hospital Collaborative Research Consortium, the Harvard Stem Cell Institute, the National Institutes of Health (grants R01HG013618, R01HL165061, F30DK135340, T32GM007753, T32GM144273), a Rappaport MGH Research Scholar Award 2024-2029, a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (CIHR), a Next Generation of Scientists Award by the Cancer Research Society, an Overseas Research Fellowship from the Japan Society for the Promotion of Science, the German Research Foundation (DFG), and a postdoctoral fellowship from the American Heart Association (25POST1377446).
Levesque and Bauer have filed a patent application (WO 2025/038842 A1) covering prime assembly technology. Pinello has financial interests in Edilytics, Inc. Pinello’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies.
