Developing PspCas13b-based enhanced RESCUE system, eRESCUE, with efficient RNA base editing

Guo Li,Xiaoxiang Hu,Jianen Gao,Xingxu Huang,Xiangyang Li,Yihan Wang,Yuzhe Wang

doi:10.1186/s12964-021-00716-z

Abstract

RNA base editing is potential for cellular function research and genetic diseases treating. There are two main RNA base editors, REPAIR and RESCUE, for in vitro use. REPAIR was developed by fusing inactivated Cas13 (dCas13) with the adenine deaminase domain of ADAR2, which efficiently performs adenosine-to-inosine (A-to-I) RNA editing. RESCUE, which performs both cytidine-to-uridine (C-to-U) and A-to-I RNA editing, was developed by fusing inactivated Cas13 (dCas13) with the evolved ADAR2. However, the relatively low editing efficiency of the RESCUE system limits its broad application. Here, we constructed an enhanced RESCUE (eRESCUE) system; this dPspCas13b-RESCUE-NES system was generated by fusing inactivated PspCas13b with the evolved ADAR2. We determined the endogenous mRNA A-to-I and C-to-U editing efficiency mediated by the dPspCas13b-RESCUE-NES system in HEK-293T cells. This new RNA base editor was then used to induce 177Ser/Gly conversion of inhibitor kappa B kinase β (IKKβ) by changing the genetic code from AGU to GGU. The results showed that the eRESCUE editor mediates more efficient A-to-I and C-to-U RNA editing than the RESCUE RNA editor, as was previously reported. The 177Ser/Gly conversion of IKKβ, accomplished by converting the genetic code from AGU to GGU, resulted in a decrease in the phosphorylation of IKKβ and downregulation of downstream IKKβ-related genes. In summary, we developed a more efficient RNA base editor, eRESCUE, which may provide a useful tool for biomedical research and genetic disease treatment.2RPHZBwwpXPmbTMUHArCFkVideo

Highlights

Previous studies have reported several tools that mediate adenosine-to-inosine (A-to-I) RNA editing in vivo [1,2,3]
There are two main RNA base editing tools in vitro: RNA editing for programmable A-to-I replacement (REPAIR) [4] and RNA editing for specific C-to-U exchange (RESCUE) [5]
Given that PspCas13b mediated the highest efficiency in knocking down endogenous KRAS compared to other optimised Cas13b systems or hairpin-mediated RNA [4], we generated mammalian codon-optimized dPspCas13b and constructed the dPspCas13b-based RESCUE system by replacing dRanCas13b with dPspCas13b to explore whether the dPspCas13b-RESCUE system could be more efficient

Summary

Introduction

Previous studies have reported several tools that mediate adenosine-to-inosine (A-to-I) RNA editing in vivo [1,2,3]. There are two main RNA base editing tools in vitro: RNA editing for programmable A-to-I replacement (REPAIR) [4] and RNA editing for specific C-to-U exchange (RESCUE) [5]. REPAIR was the first RNA base editor developed and was constructed using. We tried to improve the RNA editing efficiency of the RESCUE system by using a Cas ortholog, dPspCas13b, with a nuclear export sequence (NES) to. Li et al Cell Commun Signal (2021) 19:84 guide RNA base editors to edit mRNA in the cytoplasm. We successfully developed a PspCas13b-based enhanced RESCUE system, eRESCUE, with efficient RNA base editing abilities

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Conclusion