A circularly permuted CasRx platform for efficient, site-specific RNA editing

• Main Authors: Wang, Yuanming, Liu, Ivy Kaiwen, Liu, Mandy Mengying, Ooi, Kean Hean, Nguyen, Tram Anh, Chee, Jiunn En, Teo, Danny Shun Xiang, He, Shan, Tay, Douglas Jie Wen, Teo, Seok Yee, Liew, Kai Shin, Ge, Xiao Yu, Ng, Zhi Jian, Avagyan, Hasmik, Liu, Hao, Yi, Zirong, Chang, Keziah, Kok, Louis Eng Piew, Chen, Runjia, Yau, Chun En, Koh, Jun Wei, Wan, Yue, Tan, Meng How
• Other Authors: School of Chemistry, Chemical Engineering and Biotechnology
• Format: Article
• Language: English
• Published: 2025
• Subjects: Chemistry; RNA editing; Biochemical engineering
• Online Access: https://hdl.handle.net/10356/181953
• Institution: Nanyang Technological University

Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.