Edmonds KeHuan K, Wilkinson Max E, Strebinger Daniel, Chen Hongyu, Lash Blake, Schaefer Clarissa C, Zhu Shiyou, Liu Dangliang, Zilberzwige-Tal Shai, Ladha Alim, Walsh Michelle L, Frangieh Chris J, Vaz Reay Nicholas A, Macrae Rhiannon K, Wang Xiao, Zhang Feng
Howard Hughes Medical Institute, Cambridge, MA, USA.
Yang Tan Collective, Cambridge, MA, USA.
Nat Commun. 2025 Jul 2;16(1):6079. doi: 10.1038/s41467-025-61321-z.
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.
R2元件是一类非长末端重复（non-LTR）逆转录转座子，具有用于转基因插入的潜力。然而，实现这一目标的努力受到我们对逆转录转座子机制理解的限制。在这里，我们对来自斑胸草雀的R2（R2Tg）进行了结构和生化特征分析。我们表明，R2Tg切割其核糖体DNA靶标的两条链，并结合R2 3'UTR内的假结RNA元件以启动靶标引发的逆转录。基于这些见解指导，我们设计并表征了一种用于转基因插入的全RNA系统。我们通过消除供体上不必要的R2序列，大幅减小了系统的大小和插入疤痕。我们通过化学修饰供体RNA的5'末端并优化递送，进一步提高了整合效率，创建了一个紧凑的系统，该系统在几种人类细胞系中实现了超过80%的整合效率。这项工作扩展了基因组工程工具箱，并提供了有助于R2介导的基因插入工具未来发展的机制见解。
