All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells.

High-efficiency targeted transgene integration via primed micro-homologues.

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.