Reprogramming site-specific retrotransposon activity to new DNA sites

Christopher W. Fell, Lukas Villiger, Justin Lim, Masahiro Hiraizumi, Dario Tagliaferri, Matthew T. N. Yarnall, Anderson Lee, Kaiyi Jiang, Alisan Kayabolen, Rohan N. Krajeski, Cian Schmitt-Ulms, Harsh Ramani, Sarah M. Yousef, Nathaniel Roberts, Christopher A. Vakulskas, Hiroshi Nishimasu, Omar O. Abudayyeh () and Jonathan S. Gootenberg ()
Additional contact information
Christopher W. Fell: Harvard Medical School
Lukas Villiger: Massachusetts Institute of Technology
Justin Lim: Massachusetts Institute of Technology
Masahiro Hiraizumi: The University of Tokyo
Dario Tagliaferri: Harvard Medical School
Matthew T. N. Yarnall: Massachusetts Institute of Technology
Anderson Lee: Massachusetts Institute of Technology
Kaiyi Jiang: Harvard Medical School
Alisan Kayabolen: Harvard Medical School
Rohan N. Krajeski: Massachusetts Institute of Technology
Cian Schmitt-Ulms: Harvard Medical School
Harsh Ramani: Harvard Medical School
Sarah M. Yousef: Massachusetts Institute of Technology
Nathaniel Roberts: Integrated DNA Technologies
Christopher A. Vakulskas: Integrated DNA Technologies
Hiroshi Nishimasu: The University of Tokyo
Omar O. Abudayyeh: Harvard Medical School
Jonathan S. Gootenberg: Harvard Medical School

Nature, 2025, vol. 642, issue 8069, 1080-1089

Abstract: Abstract Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes1–6. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, 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 the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by 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 orthologues, we select an orthologue, 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 through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use 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.

Date: 2025
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DOI: 10.1038/s41586-025-08877-4

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