Researchers at UMass Chan Medical School have developed a gene editing technology that allows for the precise and efficient insertion of very large DNA segments into the human genome. Christened "prime assembly" by the study authors due to its combination of two DNA editing and assembling technologies—prime editing and Gibson assembly—this development has the potential to treat genetic diseases by replacing entire genes harboring hundreds of mutations among patient populations. Prime editing entails inserting or replacing segments of DNA in the genome. Gibson assembly is used in the lab to join multiple DNA fragments in a single test-tube reaction.
With modern gene editing technologies, such as prime editing, base editing or CRISPR-Cas9, researchers can insert or replace short segments of DNA in the genome, said Erik Sontheimer, PhD, the Pillar Chair in Biomedical Research, and vice chair and professor of RNA therapeutics. This is the equivalent of replacing a single letter or word in a book.
"Prime assembly is different. It allows us to insert longer segments of genetic code into the genome; potentially gene-length sequences. It's like replacing an entire paragraph or chapter in our genetic book," said Wen Xue, PhD, professor of RNA therapeutics.
Published in Nature , the study includes contributions from Scot A. Wolfe, PhD, professor of molecular, cell & cancer biology; Haoyang Cheng, a PhD student in the Sontheimer lab; and Jenny Gao, an MD/PhD student in the Xue lab; along with Bin Liu, PhD, a former postdoctoral researcher in the Sontheimer and Xue labs and now assistant professor of biological chemistry and pharmacology at The Ohio State University College of Medicine.
The goal of gene therapy, said Sontheimer, is to have a single technology that can replace most or all disease-causing mutations in a patient population.
Traditional gene editing technologies such as CRISPR and prime editing, however, cannot efficiently insert sequences beyond a few dozen base pairs, which is not enough to treat large genes or target multiple mutations across even moderately sized genes. In many disease cases, it also requires the development of gene editing treatments tuned specifically to the location of each patient's specific mutation. Unfortunately, for genetic diseases with dozens of different mutations between patients, this is not efficient. It would require hundreds of edits, each with its own treatment and regulatory approval. Prime assembly allows researchers to insert DNA sequences as large as 11,000 base pairs.
To achieve longer sequence replacement, researchers used a twin prime editing method to generate programmable flaps on the target DNA that introduces the DNA insertion into the genome. These flaps complement the ends of the donor DNA. This capability avoids double-strand breaks, which are prone to error and can result in unpredictable inserts or deletions in the genome. Prime assembly insertion, in contrast, creates single-strand DNA "nicks," which are considered less likely to be detrimental to the cell.
"This is a substantial step forward, not just because of the length of the DNA sequences we were able to insert but because of how relatively streamlined the process is compared to other technologies that can do large insertions," he said.
Because the prime assembly apparatus works in nondividing cells, it also has the advantage of being applicable to cells that don't divide or divide less frequently, such as neurons. Another advantage of prime assembly is its ability to stitch together multiple DNA strands to make even longer sequences, like how Gibson assembly is used in the lab to join multiple DNA fragments in a single test-tube reaction.
Moving forward, Sontheimer, Xue and colleagues are interested in elucidating the native cellular processes prime assembly takes advantage of to stitch together DNA, as well as translating these discoveries to animal models.
About UMass Chan Medical School
UMass Chan Medical School, one of five campuses of the University of Massachusetts system, comprises the T.H. Chan School of Medicine; the Morningside Graduate School of Biomedical Sciences; the Tan Chingfen Graduate School of Nursing; ForHealth Consulting at UMass Chan Medical School, a public service health care consulting division; and MassBiologics, the only nonprofit, FDA-licensed manufacturer of vaccines, biologics and viral vector gene therapies in the United States; and a thriving Nobel-Prize-winning biomedical research enterprise.
UMass Chan is advancing together to improve the health and wellness of our diverse communities throughout Massachusetts and across the world by leading and innovating in education, research, health care delivery and public service. UMass Chan has built a reputation as a world-class destination for biomedical research. It is ranked among the best medical schools in the nation for primary care education and biomedical research by U.S. News & World Report. In 2021, the Medical School received a $175 million gift from The Morningside Foundation and was renamed UMass Chan Medical School. Learn more at www.umassmed.edu .
