{Reference Type}: Journal Article
{Title}: All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.
{Author}: Chen Y;Luo S;Hu Y;Mao B;Wang X;Lu Z;Shan Q;Zhang J;Wang S;Feng G;Wang C;Liang C;Tang N;Niu R;Wang J;Han J;Yang N;Wang H;Zhou Q;Li W;
{Journal}: Cell
{Volume}: 0
{Issue}: 0
{Year}: 2024 Jul 1
{Factor}: 66.85
{DOI}: 10.1016/j.cell.2024.06.020
{Abstract}: 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.