Expanding RNA editing toolkit using an IDR-based strategy

• Published in: bioRxiv
• Main Authors: Di, Minghui, Lv, Junjun, Jing, Zhengyu, Dickinson, Bryan, Tian Chi
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 23.12.2023; Cold Spring Harbor Laboratory
• Edition: 1.1
• Subjects: Editing; Fusion protein; Histidine; Immunogenicity; Molecular Biology; RNA editing
• ISSN: 2692-8205; 2692-8205
• DOI: 10.1101/2023.12.23.573192

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 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.Competing Interest StatementB.C.D. have a patent filed for the CIRTS technology. B.C.D. is a founder and holds equity in Tornado Bio, Inc., a company developing RNA-programmable therapies.