The
development of programmable, site-specific gene editing tools has
provided methods and strategies for correcting pathogenic mutations,
such as emerging RNA editing technologies. Unlike DNA editing, RNA
editing transiently modifies RNA transcripts, thereby avoiding the
long-term safety risks associated with DNA off-target effects.
Two adenosine deaminase ADAR (adenosine deaminase acting on RNA)-derived RNA adenosine-to-inosine (A-to-I) editing systems have been reported. The first system utilizes exogenous ADAR fused with locator proteins and directed by guide RNA to induce editing at target sites. However, the high expression of exogenous ADAR proteins can lead to severe transcriptome-wide off-target effects. The second system recruits endogenous ADAR proteins to mediate RNA editing at target sites. While this approach avoids off-target effects, the sequence preferences of endogenous ADAR and its variable expression levels across tissues limit its editing scope and efficiency. Therefore, developing an RNA editing system with low off-target activity and broad editing applicability is of significant importance for RNA research and therapeutic applications.
Recently, a collaborative team led by Professor Chen Jia from
the School of Life Science and Technology (SLST) at ShanghaiTech
University and Professor Yang Li at Fudan University published a study
titled “Specific and Efficient RNA A-to-I Editing through Cleavage of an ADAR Inhibitor” () in Nature Biotechnology.
The study reports a highly efficient, precise, and broadly applicable
RNA adenosine base editing system called RtABE (RNA transformer
Adenosine Base Editor).
First, the collaborative team screened protein domains capable of inhibiting the ADAR2 deaminase domain (ADAR2DD). They discovered that certain deoxycytidine deaminase inhibitor (dCDI) domains from the APOBEC family of cytidine deaminases could act as ADAR inhibitors (ADIs). In the RtABE system, the ADI derived from human APOBEC3 (A3DADI) was fused with ADAR2DD to suppress its deaminase activity. At off-target sites, ADAR2DD remains inactive due to the A3DADI fusion. At target sites, engineered ADAR guide RNA (eagRNA) recruits split TEV protease and the ADAR2DD-A3DADI fusion protein, cleaving the A3DADI to activate ADAR2DD for site-specific editing. To enhance precision, protein engineering was used to generate two ADAR2DD mutants (K475Q+E488Q and K475I+S486A), leading to the development of the RtABE_V1 and RtABE_V2 systems. RtABE significantly reduced transcriptome-wide off-target effects while maintaining high editing efficiency at target sites.
Figure 1: Schematic of the RtABE working mechanism
Compared to RNA editors that rely on engineered RNAs to recruit endogenous ADAR, RtABE achieved highly efficient editing across broader sequence contexts (e.g., UAN, AAN, CAN, GAN). Furthermore, the team delivered RtABE via a single adeno-associated virus (AAV) into Hurler syndrome (mucopolysaccharidosis type I) model mice. RtABE demonstrated sustained, efficient, and precise RNA A-to-I editing in vivo, with no significant off-target editing detected in the liver of treated mice. Thus, RtABE represents a precise and efficient RNA base editing system with wide applicability.
Figure 2: Comparison of editing efficacy between RtABE_V2 and MCP-ADAR2DD systems. (Left) Editing efficiencies induced by RtABE_V2 and MCP-ADAR2DD in mouse liver. (Right) Transcriptome-wide off-target counts induced by RtABE_V2 and MCP-ADAR2DD in mouse liver.
Based on the collaborative team’s findings in disease model mice, RtABE offers new hope for potential clinical applications in treating genetic disorders and other severe human diseases.
Professors Chen Jia and Yang Li are the corresponding authors. SLST PhD students Li Guangye and Chen Guo in Chen Jia’s group, PhD student Yuan Guohua from Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, and Wei Jia from Children’s Hospital of Fudan University are the co-first authors. ShanghaiTech is the primary affiliation.
