New Genome-Editing Method Achieves Megabase-Scale DNA Integration

Current genome editing approaches face real constraints: they generally either rely on untargeted gene delivery or on short DNA edits that must be tailored to each patient’s specific mutation. A new paper published today in Nature describes a genome engineering method called prime assembly that uses CRISPR-targeted dual flap synthesis to assemble and integrate DNA sequences at specific locations within human cells, an approach the researchers used to perform targeted exon recoding, transgene integration and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells.

Prime assembly builds on prime editing, a technology already capable of making small, precise insertions, deletions and base swaps. Because many genetic diseases stem from hundreds of distinct mutations, correcting them with prime editing alone can require a different edit for nearly every patient. Prime assembly instead aims to fix almost any mutation within a gene using a single approach, enabling RNA-programmable, site-specific integration of single- or double-stranded DNA fragments ranging from medium to large in size.

The method works in one step, writing new DNA “flaps” into specific genomic locations. These flaps act as tethers that grab onto DNA fragments with matching ends, and the resulting insert, which can consist of one or more gene-sized pieces of DNA, becomes a large, permanent edit. “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 Daniel Bauer from Boston Children’s Hospital and co-senior author of the study. “Because the method is based on prime editing, it is much less likely to cause off-target effects compared to other gene editing methods.”

That distinction matters because untargeted insertion methods carry the risk of activating the wrong genes in the wrong context, potentially leading to cancerous outcomes, a risk prime assembly’s targeted insertion approach avoids. The method also does not depend on DNA double strand breaks or double strand donors, both of which can be toxic and stress cells. And whereas homology-directed repair depends on cell cycle progression and is largely restricted to the rare, more vulnerable dividing cells in the body, prime assembly is similarly active in both dividing and non-dividing cells.

The team’s next steps involve digging further into the molecular mechanisms behind the method to make it more efficient and precise, with an eye toward eventual clinical use. Because it can correct multiple mutations through a single approach, the researchers see potential for more broadly applicable treatments for 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,” Bauer said. “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.”