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. 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.

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 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.