RESUMO
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. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.