Schematic illustrating the mechanism of target-primed reverse transcription (TPRT) used by non-long terminal repeat (nLTR) retrotransposons for insertion. The process involves initial DNA nicking, use of the nicked strand to prime reverse transcription of the retrotransposon RNA (payload), and eventual integration. Source: Excerpted from Figure 1 in the paper from Fell, C.W., Villiger, L., Lim, J. et al. Nature (2025). https://doi.org/10.1038/s41586-025-08877-4
A novel gene editing tool named STITCHR, developed at Mass General Brigham and collaborating institutions, overcomes central limitations of current technologies by enabling the scarless insertion of entire genes or large DNA fragments up to 12.7 kb. Based on reprogrammed retrotransposon activity guided by a Cas9 nickase and using RNA templates, STITCHR shows potential for developing “one-and-done” therapies for genetic diseases involving multiple mutations or requiring whole-gene replacement. In addition, its mechanism avoids double-strand DNA breaks, and its reliance solely on RNA templates may simplify therapeutic delivery strategies. A paper on the research has been published in Nature.
Co-senior author Omar Abudayyeh, PhD, an investigator at the Gene and Cell Therapy Institute (GCTI) at Mass General Brigham highlighted that CRISPR has transformed the field but added that it has limitations. “CRISPR can’t target every location in the genome, and it can’t fix the thousands of mutations present in diseases like cystic fibrosis,” Abudayyeh said in a release. “When we started our lab, one of the big things we wanted to figure out was how to insert large pieces of genes, or even entire genes, to replace faulty ones. This would allow us to target every mutation for a disease with a single gene editing construct.”
The development process began with a comprehensive computational search across animal genomes. It eventually uncovers thousands of site-specific non-long terminal repeat (nLTR) retrotransposons and reveals diverse, previously unknown insertion preferences and potential retargeting mechanisms. Profiling selected candidates biochemically and in mammalian cells identified an R2 retrotransposon from the zebra finch (R2Tg) as active. They also found that the cells were capable of being reprogrammed for scarless insertion at new genomic sites using engineered RNA payloads. To boost efficiency, the researchers fused R2Tg to a CRISPR-Cas9 nickase (SpCas9H840A). Further screening pinpointed another orthologue, R2Tocc, that had favorable characteristics like natural reprogrammability and minimal activity at its native 28S target site. Engineering a fusion of this optimized retrotransposon with the Cas9 nickase resulted in the final system, dubbed STITCHR (site-specific target-primed insertion through targeted CRISPR homing of retroelements).
The researchers reported that the STITCHR platform proved highly versatile, successfully demonstrating scarless edits ranging from single base changes up to previously mentioned kilobase sizes. It has potential for a range of uses, including gene replacement, using in vitro transcribed or synthetic RNA templates, and showing activity in non-dividing cells.
Filed Under: Biospecimens, Cell & gene therapy
