Chen Pingbo, Li Xiangyang, Zhou Qian, Chen Jingzhou, Lu Lijin, Wang Pei, Zhang Guiquan, Sun Dongxiao, Huang Xingxu, Liu Jianghuai, Wang Xiaolong
Hainan Institute of Northwest A&F University, Sanya 572025, China.
International Joint Agriculture Research Center for Animal Bio-Breeding of Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf522.
The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by re-configuring guide/template RNAs to drive prime edits into the target strand (TS), instead of the conventional non-TS. We first devise a dual-RNA uPEn strategy by supplementing a cleavage-competent sgRNA with an accessory template RNA for modifying target strand (ActRNA:t). Characterization of the dual-RNA system allows us to next develop a bifunctional target strand-programming pegRNA (tsp-pegRNA). Both the dual- and single-RNA upstream-modifying uPEn forms (versions 3.1/3.2) successfully drive diverse types of accurate edits into a panel of locations refractory to the standard uPEn and the latest nickase PE. Moreover, we provide insights on the role of uPEn's helper module (i.e. i53) in driving TS prime edits. Additional co-administration of a DNA-dependent protein kinase inhibitor with uPEn3.2 leads to further optimization of editing purities. Together, these advances transform uPEn into a highly applicable tool with much-expanded editable space, and lay a strong foundation for future development of PEn/PE platforms.
核酸酶引导编辑器（PEn）将双链断裂（DSB）诱导与逆转录相结合以进行编辑。最近，通过同时应用DNA修复调节剂开发出了高活性的PEn形式（如uPEn）。虽然标准的uPEn仅在核酸酶诱导的DNA断裂下游引入编辑，但我们寻求创新设计，通过重新配置引导/模板RNA，将引导编辑驱动到目标链（TS）中，而不是传统的非目标链，从而实现上游定向编辑。我们首先设计了一种双RNA的uPEn策略，通过用用于修饰目标链的辅助模板RNA（ActRNA:t）补充具有切割能力的sgRNA。对双RNA系统的表征使我们能够进一步开发一种双功能的目标链编程pegRNA（tsp-pegRNA）。双RNA和单RNA上游修饰的uPEn形式（3.1/3.2版本）都成功地在一组对标准uPEn和最新的切口酶PE难以处理的位置上驱动了多种类型的精确编辑。此外，我们还深入了解了uPEn的辅助模块（即i53）在驱动TS引导编辑中的作用。将DNA依赖性蛋白激酶抑制剂与uPEn3.2共同给药可进一步优化编辑纯度。总之，这些进展将uPEn转变为一种具有更广泛可编辑空间的高度适用工具，并为PEn/PE平台的未来发展奠定了坚实基础。
