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.

失活的 Cas13 直系同源物已与 C 端的突变人 ADAR2 脱氨酶结构域融合，以便在选定的转录本中实现可编程的腺苷-肌苷 （A 到 I） RNA 编辑。尽管前景广阔，但现有的 RNA 编辑工具通常在功效和特异性之间进行权衡，脱靶编辑仍然是一个未解决的问题。在这里，我们描述了通过理性蛋白质工程、基于 CasRx 的 RNA 可编程编辑技术 （xPERT） 开发优化的 RNA 编辑平台。我们证明，通过环状排列对 CasRx K940L 突变体进行拓扑重排，从而为脱氨酶结构域的栓系提供稳健的支架。我们将我们的工具与 REPAIR 系统进行基准测试，结果表明 xPERT 表现出像 REPAIRv1 一样强大的目标活动，但像 REPAIRv2 一样表现出较低的脱靶编辑。我们的 xPERT 平台可用于改变 RNA 序列信息，而不会有基因组损伤的风险，影响暂时的细胞变化和定制蛋白质功能。