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RNA base editors should ideally be free of immunogenicity, compact, efficient, and specific, which has not been achieved for C > U editing. Here we first describe a compact C > U editor entirely of human origin, created by fusing the human C > U editing enzyme RESCUE-S to Cas inspired RNA targeting system (CIRTS), a tiny, human-originated programmable RNA-binding domain. This editor, CIRTS-RESCUEv1 (V1), was inefficient. Remarkably, a short histidine-rich domain (HRD), which is derived from the internal disordered region (IDR) in the human CYCT1, a protein capable of liquid-liquid phase separation (LLPS), enhanced V1 editing at on-targets as well as off-targets, the latter effect being minor. The V1-HRD fusion protein formed puncta characteristic of LLPS, and various other IDRs (but not an LLPS-impaired mutant) could replace HRD to effectively induce puncta and potentiate V1, suggesting that the diverse domains acted via a common, LLPS-based mechanism. Importantly, the HRD fusion strategy was applicable to various other types of C > U RNA editors. Our study expands the RNA editing toolbox and showcases a general method for stimulating C > U RNA base editors.

Catalytically inactive CRISPR‐Cas13 (dCas13)‐based base editors can achieve the conversion of adenine‐to‐inosine (A‐to‐I) or cytidine‐to‐uridine (C‐to‐U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199‐amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre‐crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A‐to‐I and C‐to‐U base editing with low transcriptome off‐target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases. [ABSTRACT FROM AUTHOR]

Abstract Catalytically inactive CRISPR‐Cas13 (dCas13)‐based base editors can achieve the conversion of adenine‐to‐inosine (A‐to‐I) or cytidine‐to‐uridine (C‐to‐U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199‐amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre‐crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A‐to‐I and C‐to‐U base editing with low transcriptome off‐target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases.

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. Video Abstract

Abstract The currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity. Video 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. 2RPHZBwwpXPmbTMUHArCFk Video Abstract [ABSTRACT FROM AUTHOR]

The currently available RESCUE RNA base editing system demonstrates considerable potential for the treatment of genetic diseases at the transcriptional level. However, the relatively high incidence of off-target events hampers the precise RNA editing, thereby limiting its use in the clinical setting. This study describes a new RNA base editing method, named ecRESCUE, which utilizes inducible stabilization of the protein ecDHFR DD fused at the C-terminal of the original RESCUE system. In vitro experiments in 293T cells showed that the ecRESCUE editor markedly reduced the incidence of off-target single nucleotide polymorphisms without affecting the RNA A-to-I and C-to-U base editing efficiency. Altogether, these results demonstrate that the inducible ecRESCUE system represents an attractive approach to regulate and improve the outcome of the available RNA base editor with reduced off-targeting activity. AehByCFVFypSuirAdhmseq Video Abstract [ABSTRACT FROM AUTHOR]