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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., Competing Interests: B.C.D. has 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., (© 2024 The Author(s).)

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOF Q829X , resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized Otof Q829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOF Q829X (emxABE-T) administered during the postnatal day 0-3 period in Otof Q829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 Otof Q829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOF Q829X -induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons., Competing Interests: Declaration of interests Xing Wang disclosed a patent application related to this work. L.S. and Y.X. disclosed a patent application related to the AAV vector used in this work. H.Y. is the cofounder of HuidaGene Therapeutics Co., Ltd. The remaining authors declare no competing interests., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)