Scientists can already edit individual "letters" of DNA. It is far more difficult to replace a large stretch of a gene at once — especially without a double-strand break in the DNA.
A new paper in the journal Nature describes prime assembly — a method that extends the capabilities of prime editing and makes it possible to insert DNA fragments several thousand nucleotides long into designated regions of the genome.
The system creates short single-stranded 3′ "tails" on the two DNA strands. These are designed to match the ends of the donor fragment. After that, the cell's own machinery completes and joins the molecule.
In principle, this resembles Gibson assembly — a way of joining DNA fragments in a test tube. Only here, such assembly takes place directly inside a living cell.
In one of the experiments, the researchers worked with the TINF2 gene, mutations in which are associated with severe disorders of telomere maintenance. They replaced a stretch of 106 nucleotides while preserving the amino acid sequence of the protein. In K562 cells, editing efficiency reached 33,1%.
The authors also demonstrated the integration of donor sequences up to 12,1 kilobases in size and the possibility of large rearrangements of an already existing genome, up to the megabase scale.
Particularly important is that prime assembly worked not only in laboratory cell lines but also in primary human CD34+ hematopoietic cells and T lymphocytes, including non-dividing cells. Efficiency there is still considerably lower, but the very fact of such editing broadens the potential scope of the method.
Also interesting is the idea of a therapy that does not depend on a specific mutation. Instead of creating a separate tool for each mutation, one could try to replace or recode an entire problematic region of a gene at once.
That is, the principle may change: one mutation → one correction per gene region → one universal construct.
For now, prime assembly remains an experimental technology. It has yet to be made more efficient in therapeutically important cells and to solve the problem of delivering the components directly into the body.
But the concept itself shows an important shift in genetic engineering: genome editing is gradually moving from cutting DNA to its programmable assembly inside the cell.
