New prime assembly method enables long and precise DNA insertions

Genomic editing holds great potential yet continues to have its limitations. Most current methods either rely on untargeted gene delivery or short DNA edits that need to be individualized to each patient. A new paper published today in Nature describes a novel genome engineering method called prime assembly that allows long DNA fragments to be stitched into precise and programmable target positions within living cells. This approach may allow for the development of universal gene therapies that can apply to many patients.

Prime assembly builds upon the techniques of prime editing, an advanced technology that allows for precise yet small insertions, deletions, and base swaps. Because many genetic diseases involve hundreds of different mutations, current gene editing strategies for a disease might require many different edits to achieve each needed small change. On the other hand, prime assembly could correct the DNA with a single approach that could fix almost any mutation in the gene.

Prime assembly's single step process writes in new DNA flaps to specific locations in the genome. The flaps serve as tethers designed to grab onto DNA fragments with matching ends. The precisely assembled DNA inserts, which can be one or more gene-sized DNA pieces, become 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. Because the method is based on prime editing, it is much less likely to cause off-target effects compared to other gene editing methods."

Daniel Bauer, MD, PhD, Director of the Gene Therapy Program, Boston Children's Hospital and co-senior author

One such off-target effect is the potential for toxicity. Untargeted insertion methods can turn on the wrong genes in the wrong context, leading to potentially cancerous outcomes, whereas 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 are rare in the body and more susceptible to unwanted DNA changes, prime assembly works in nondividing cells.

Building on this exciting milestone, the team next is looking to further investigate the molecular mechanisms which would allow them to engineer even more efficient and precise systems. As they fine tune their approach, they hope this technology will have downstream impact in the clinic. With the ability to correct multiple mutations at once, this technology could perhaps lead to generalizable solutions for treating genetic disorders.

"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. "We're also exploring a number of applications of prime assembly to deliver genetic payloads as mutation-agnostic therapies to restore gene control for devastating inherited human diseases with unmet clinical need."