Existing C-to-U RNA editing tools face several challenges including the risk of concurrent DNA mutations caused by natural APOBEC family, and the large-size anchoring domains associated with immunogenicity due to microbial origin of Cas13 protein, which restricts their potential for clinical translation.

In a study published in PNAS, a team led by HAO Pei from the Shanghai Institute of Materia Medica of the Chinese Academy of Sciences (CAS) and LI Xuan from the Center for Excellence in Molecular Plant Sciences of CAS developed a novel C-to-U RNA editing tool, ADAR2-mimic C-to-U base editor for RNA (AMBER) which exhibits high editing efficiency, low immunogenicity and minimal off-target effects at the transcriptome level.

Researchers introduced 17 key amino acid substitutions previously reported in the RESCUE-S editing system into the catalytic domain of ADAR2. AMBER used the native double-stranded RNA binding domain of ADAR2 for target recognition. This design generated a fully human-derived C-to-U RNA editing system that does not rely on bacterial Cas proteins and is expected to have lower immunogenicity.

AMBER was tested with a reporter based on a mutant enhanced green fluorescent protein (eGFP). With the help of a designed guide RNA, AMBER introduced on-target C-to-U editing on the eGFP transcript, and restored the correct codon and recovered eGFP fluorescence. These results confirmed that ADAR2 can be reprogrammed into a sequence-specific C-to-U RNA deaminase.

Moreover, AMBER was evaluated across multiple endogenous targets and exogenous disease-related mutant targets in different cell lines. Researchers optimized the guide RNA design including length, the sequence context around the target cytidine, and strategies to reduce bystander editing in the editing window. They established design principles that support high-efficiency and high-precision C-to-U editing while minimizing off-target events.

To assess AMBER’s therapeutic potential in vivo, researchers delivered DNA constructs encoding AMBER into mice via tail-vein injection. Bioluminescence imaging of a Firefly luciferase reporter co-expressed with AMBER indicated that the editor accumulated mainly in the liver. RNA sequencing of liver tissue showed robust C-to-U editing at intended RNA targets. 

Meanwhile, transcriptome-wide analysis revealed minimal off-target editing events, and that global gene expression was minimally affected when compared with the control mice. These findings indicated that AMBER can perform efficient and relatively specific RNA editing in vivo.

AMBER represents a new class of human-derived C-to-U RNA base editors. It emulates the mode of natural adenosine deaminase acting on RNA (ADAR) and achieves precise C-to-U editing of target RNAs in cultured cells and mouse liver. This study suggests that AMBER could be a platform for developing RNA editing therapies for genetic diseases caused by pathogenic T-to-C mutations.