1. A circularly permuted CasRx platform for efficient, site-specific RNA editing.
- Author
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Wang Y, Liu KI, Liu MM, Ooi KH, Nguyen TA, Chee JE, Teo SXD, He S, Tay JWD, Teo SY, Liew KS, Ge XY, Ng ZJ, Avagyan H, Liu H, Yi Z, Chang K, Kok EPL, Chen R, Yau CE, Koh JW, Wan Y, and Tan MH
- Abstract
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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2024
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