Retargeted retrotransposons insert multi-kilobase cargo at new sites

Site-specific retroelements, such as R2 retrotransposons, have potential as programmable genome-editing systems, but so far have only been used for targeted insertion at genomic safe-harbor loci. In a paper now published in Nature, Fell et al. profile the evolution of site-specific retrotransposon families to gain insight into target site preferences, which they then used to engineer reprogramming.

By surveying 4,464 animal assemblies derived from Genbank, the authors discovered several new site-specific retrotransposon families, including families with multiple integration preferences and with differing 5′ and 3′ site predictions, which might indicate retargeting mechanisms. They zoned in on a retrotransposon from the zebra finch (Taeniopygia guttata), called R2Tg, for biochemical profiling and characterized its activity in mammalian cells, which showed that R2Tg can insert payloads by reverse transcription of RNA and nicking of genomic DNA. Notably, the authors found that R2Tg can be retargeted by engineering the payload, which allowed scarless insertion of the payload at new genomic sites. The activity of the reprogrammed R2Tg was enhanced by fusing it with CRISPR–Cas9 nickase.