Expanding RNA editing toolkit using an IDR-based strategy

Minghui Di; Junjun Lv; Zhengyu Jing; Yijie Yang; Kunlun Yan; Jianguo Wu; Jianyang Ge; Simone Rauch; Bryan C Dickinson; Tian Chi

doi:10.1016/j.omtn.2024.102190

Expanding RNA editing toolkit using an IDR-based strategy

Mol Ther Nucleic Acids. 2024 Apr 16;35(2):102190. doi: 10.1016/j.omtn.2024.102190. eCollection 2024 Jun 11.

Authors

Minghui Di^{1

2}, Junjun Lv^{1

2}, Zhengyu Jing^{1

2}, Yijie Yang¹, Kunlun Yan¹, Jianguo Wu^{1

2}, Jianyang Ge^{1

2}, Simone Rauch³, Bryan C Dickinson³, Tian Chi^{1

4}

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
³ Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
⁴ Department of Immunobiology, Yale University Medical School, New Haven, CT, USA.

Abstract

RNA base editors should ideally be free of immunogenicity, compact, efficient, and specific, which has not been achieved for C > U editing. Here we first describe a compact C > U editor entirely of human origin, created by fusing the human C > U editing enzyme RESCUE-S to Cas inspired RNA targeting system (CIRTS), a tiny, human-originated programmable RNA-binding domain. This editor, CIRTS-RESCUEv1 (V1), was inefficient. Remarkably, a short histidine-rich domain (HRD), which is derived from the internal disordered region (IDR) in the human CYCT1, a protein capable of liquid-liquid phase separation (LLPS), enhanced V1 editing at on-targets as well as off-targets, the latter effect being minor. The V1-HRD fusion protein formed puncta characteristic of LLPS, and various other IDRs (but not an LLPS-impaired mutant) could replace HRD to effectively induce puncta and potentiate V1, suggesting that the diverse domains acted via a common, LLPS-based mechanism. Importantly, the HRD fusion strategy was applicable to various other types of C > U RNA editors. Our study expands the RNA editing toolbox and showcases a general method for stimulating C > U RNA base editors.

Keywords: CIRTS; IDR; LLPS; MT: RNA/DNA Editing; RNA base editing; compact; human origin; immunogenicity.