Li Guangye,Chen Guo,Yuan Guo-Hua,Wei Jia,Ni Qingyang,Wu Jing,Yang Bei,Yang Li,Chen Jia
RNA editing can be a promising therapeutic approach. However, ectopic expression of RNA editing enzymes has been shown to trigger off-target editing. Here we identified adenosine deaminase acting on RNA (ADAR) inhibitors (ADIs) that suppress the activity of the fused ADAR2 deamination domain (ADAR2 DD ). Using these specific ADIs, we develop an RNA transformer adenosine base editor (RtABE) with high specificity. Fusing ADI to ADAR2 DD , RtABE remains inactive until it binds to its target site. After binding to the target site, ADI is cleaved from ADAR2 DD , and RtABE becomes active. RtABE can induce efficient editing in broad sequence contexts, including UAN, AAN, CAN and GAN. Using an adeno-associated virus for delivery of RtABE enables therapeutic RNA correction and restoration of α- l -iduronidase activity in Hurler syndrome mice with no substantial off-target editing. RtABE is a specific and efficient RNA editing system with a broad scope that may be a better alternative to existing RNA editing tools.
Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.
Aging impairs intestinal stem cell (ISC) function, disrupting epithelial homeostasis and regenerative repair. Loss of ISC quiescence promotes intestinal dysfunction and contributes to organismal aging. Here, we report an epitranscriptomic mechanism through which a decrease in adenosine-to-inosine (A-to-I) RNA editing by the adenosine deaminase ADAR in ISCs during aging disrupts a conserved signaling axis that maintains ISC quiescence. In Drosophila melanogaster , ISC-specific loss of dADAR triggered hyperproliferation and tissue aging, whereas dADAR overexpression reduced age-related gut dysfunction and extended life span. Intestinal injury induced a decrease in dADAR, which was necessary for tissue regeneration, and in the zinc finger protein dZn72D, which stabilized dADAR to support intestinal homeostasis during aging. dADAR edited transcripts encoding the RNA binding protein Pumilio (dPUM) to generate an isoform that inhibited translation of two components of the mitogen-activated protein kinase (MAPK), dEGFR and dERK, thereby maintaining ISC quiescence. The abundance of ADAR2 and of the dZn72D ortholog ZFR decreased in human colonic crypts with aging and in mouse colon after injury, suggesting that attenuation of ADAR activity may play a conserved role in ISC aging and in facilitating colonic regeneration. Our work identifies a conserved mechanism of epitranscriptomic regulation that safeguards tissues and the decay of ADAR-mediated RNA editing as a form of age-related epigenetic information loss that disrupts ISC homeostasis.
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