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

Type VI CRISPR-Cas systems have recently been harnessed as tools for transcriptome engineering in mammalian cells. Specifically, inactive Cas13 orthologs have been fused at the C-terminus to a mutant human ADAR2 deaminase domain to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing tools generally suffer from a trade-off between efficacy and specificity and trans off-target edits remain an unsolved problem. Here, we describe the development of an optimized RNA editing platform by rational protein engineering. We demonstrate that topological rearrangement of a CasRx mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. Notably, we benchmark our tool against other methods1 and show that our CasRx-based Programmable Editing of RNA Technology (xPERT) exhibits high on-target activity like REPAIRv1 and almost no trans off-target editing like REPAIRv2. Our rationally engineered CasRx protein scaffold may be deployed in other transcriptome engineering applications.