Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons
Abstract R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61321-z |
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| author | KeHuan K. Edmonds Max E. Wilkinson Daniel Strebinger Hongyu Chen Blake Lash Clarissa C. Schaefer Shiyou Zhu Dangliang Liu Shai Zilberzwige-Tal Alim Ladha Michelle L. Walsh Chris J. Frangieh Nicholas A. Vaz Reay Rhiannon K. Macrae Xiao Wang Feng Zhang |
| author_facet | KeHuan K. Edmonds Max E. Wilkinson Daniel Strebinger Hongyu Chen Blake Lash Clarissa C. Schaefer Shiyou Zhu Dangliang Liu Shai Zilberzwige-Tal Alim Ladha Michelle L. Walsh Chris J. Frangieh Nicholas A. Vaz Reay Rhiannon K. Macrae Xiao Wang Feng Zhang |
| author_sort | KeHuan K. Edmonds |
| collection | DOAJ |
| description | Abstract R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools. |
| format | Article |
| id | doaj-art-427648de425a4d4bb2efa899c6667494 |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-427648de425a4d4bb2efa899c66674942025-08-20T03:03:44ZengNature PortfolioNature Communications2041-17232025-07-0116112010.1038/s41467-025-61321-zStructure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposonsKeHuan K. Edmonds0Max E. Wilkinson1Daniel Strebinger2Hongyu Chen3Blake Lash4Clarissa C. Schaefer5Shiyou Zhu6Dangliang Liu7Shai Zilberzwige-Tal8Alim Ladha9Michelle L. Walsh10Chris J. Frangieh11Nicholas A. Vaz Reay12Rhiannon K. Macrae13Xiao Wang14Feng Zhang15Howard Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteBroad Institute of MIT and HarvardHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteBroad Institute of MIT and HarvardHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteBroad Institute of MIT and HarvardHoward Hughes Medical InstituteAbstract R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools.https://doi.org/10.1038/s41467-025-61321-z |
| spellingShingle | KeHuan K. Edmonds Max E. Wilkinson Daniel Strebinger Hongyu Chen Blake Lash Clarissa C. Schaefer Shiyou Zhu Dangliang Liu Shai Zilberzwige-Tal Alim Ladha Michelle L. Walsh Chris J. Frangieh Nicholas A. Vaz Reay Rhiannon K. Macrae Xiao Wang Feng Zhang Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons Nature Communications |
| title | Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons |
| title_full | Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons |
| title_fullStr | Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons |
| title_full_unstemmed | Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons |
| title_short | Structure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons |
| title_sort | structure and biochemistry guided engineering of an all rna system for dna insertion with r2 retrotransposons |
| url | https://doi.org/10.1038/s41467-025-61321-z |
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