Expanding RNA editing toolkit using an IDR-based strategy

• Published in: Molecular therapy. Nucleic acids Vol. 35; no. 2; p. 102190
• Main Authors: Di, Minghui, Lv, Junjun, Jing, Zhengyu, Yang, Yijie, Yan, Kunlun, Wu, Jianguo, Ge, Jianyang, Rauch, Simone, Dickinson, Bryan C., Chi, Tian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.06.2024; American Society of Gene & Cell Therapy; Elsevier
• Subjects: CIRTS; compact; human origin; IDR; immunogenicity; LLPS; MT: RNA/DNA Editing; Original; RNA base editing; LLPS; MT: RNA/DNA Editing; compact; IDR; human origin; CIRTS; RNA base editing; immunogenicity
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2024.102190

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. [Display omitted] Chi and colleagues show that protein regions capable of inducing liquid-liquid phase separation can be harnessed to potentiate various types of C > U RNA base editors, offering a simple and general strategy for their optimization.