Prime assembly enables large-scale, targeted DNA editing

A new genome editing method allows long DNA fragments to be integrated into precise positions within the genome of cells.

Many genome editing approaches are limited either by the size of the DNA changes they can make or by the need to tailor short edits to individual mutations. These limitations represent important obstacles, especially when addressing genetic diseases rooted in hundreds of mutations. A new approach, called prime assembly, now builds upon prime editing technology to allow long DNA fragments to be inserted into specific target positions (see BioNews 1021). Although the method is still in its early stages of development, the researchers are exploring its potential to make larger genetic changes and address multiple mutations at once.

'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,' said Professor Daniel Bauer, director of the gene therapy program at Boston Children’s Hospital, Massachusetts and co-senior author of the study published in Nature.

Prime assembly relies on creating single-stranded DNA flaps at specific locations in the genome. These flaps connect to DNA segments designed by the researchers, which can be up to 12.1 kilobases at a single site – a size large enough to encompass gene fragments. The segments become integrated into the genome as a permanent edit via a single targeted editing step.  

'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,' explained Professor Bauer.

Current genome editing approaches can carry risks from off-target effects and unintended genomic changes. For example, an off-target DNA insertion could accidentally turn on a gene in an incorrect context, leading to cancer. Additionally, some techniques rely on DNA double-strand breaks or double-stranded DNA templates, which can be highly stressful to cells. Prime assembly avoids the need to deliberately break both strands of DNA at the target site and can use single-stranded DNA. However, the researchers noted that temporary breaks to both strands can still occur during the editing process.

Researchers also showed that prime assembly can work in both dividing and non-dividing cells, and in some experiments it outperformed existing targeted integration methods. This could be therapeutically valuable because some established approaches are restricted to actively dividing cells.

The study remains preclinical, relying on experiments performed in cell lines and primary cells from healthy donors. How the technology can be delivered into cells in an organism remains to be explored, and the researchers hope to engineer more efficient and precise systems that could be applied to patient care.

'We're working to improve the delivery of the prime assembly components to disease-relevant human cells in vivo, such as haematopoietic stem cells for blood disorder therapies,' said Professor Bauer.