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TitleStructure and biochemistry-guided engineering of an all-RNA system for DNA insertion with R2 retrotransposons.
Journal, issue, pagesNat Commun, Vol. 16, Issue 1, Page 6079, Year 2025
Publish dateJul 2, 2025
AuthorsKeHuan 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 /
PubMed AbstractR2 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 ...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.
External linksNat Commun / PubMed:40603868 / PubMed Central
MethodsEM (single particle)
Resolution3.2 Å
Structure data

EMDB-47091, PDB-9dou:
Taeniopygia guttata R2 retrotransposon (R2Tg) initiating target-primed reverse transcription
Method: EM (single particle) / Resolution: 3.2 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

ChemComp-ZN:
Unknown entry

Source
  • taeniopygia guttata (zebra finch)
KeywordsRNA BINDING PROTEIN/RNA/DNA / retrotransposon / LINE / reverse transcriptase / endonuclease / RNA BINDING PROTEIN-RNA-DNA complex

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