Reprogramming site-specific retrotransposon activity to new DNA sites

Retroelements play a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat (nLTR) retrotransposons have spread widely via preferential integration into repetitive genomic sequences like microsatellite regions and ribosomal DNA genes. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Using a computational pipeline to discover multiple new site-specific retrotransposon families, we profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences, and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from Taeniopygia guttata, as an ortholog that can be retargeted via payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologs, we select an ortholog, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9H840A-R2Tocc, a system we name Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR). STITCHR enables scarless, efficient, installation of edits ranging from a single base to 12.7 kb, gene replacement, and utilization of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.