Retrons are promising gene editing tools because they can produce multi-copy single-stranded DNA in cells via self-primed reverse transcription. However, their potential for inserting genetic cargos in eukaryotes remains largely unexplored. Here we report the discovery and engineering of highly efficient retron-based gene editors for mammalian cells and vertebrates. Through bioinformatic analysis of metagenomic data and functional screening, we identified novel retron reverse transcriptases (RTs) that are highly active in mammalian cells. Rational design further improved the editing efficiency to levels comparable with conventional single-stranded oligodeoxynucleotide donors but from a genetically encoded cassette. Small molecule inhibitors of non-homologous end joining factors and Cas9-DNA repair protein fusions further increase homology-directed repair. Retron editors also exhibited robust activity with Cas12a nuclease and Cas9 nickase, expanding the genomic target scope and bypassing the need for a DNA double-stranded break. Using a rationally engineered retron editor, we incorporate a split GFP epitope tag for live cell imaging. Finally, we develop an all-RNA delivery strategy to enable DNA-free gene editing in cells and vertebrate embryos. This work establishes retron editors as a versatile and efficient tool for precise gene editing, offering new opportunities for biotechnology and biomedical research.

Discovery and Engineering of Retrons for Precise Genome Editing