🧬 PubMed RNA Editing Daily Digest

RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly read out as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.

The TadA component of adenine base editors (ABEs) induces widespread RNA off-target edits and raises safety concerns for their applications. However, the extent of RNA editing-related toxicity remains elusive, and high-throughput engineering of ABEs focusing on RNA editing activities remains challenging. Here, we demonstrate that RNA off-target editing of classical ABEs leads to substantial toxicity in vitro and in vivo. We then design a rapid, cost-effective, and sensitive fluorescent RNA inosine sensor to accelerate RNA off-target editing evaluation and high-throughput screening in mammalian cells. Deep mutation scanning with the RNA sensor identifies various TadA8e mutants displaying minimized RNA editing activity, with the representative H52L/D53R mutant compatible with both SpCas9 and the compact IscB nickase. We show that the engineered ABEs could efficiently target clinically relevant sites in vitro and in vivo with enhanced precision, thereby providing promising tools for applications in which RNA editing-related toxicity should be carefully evaluated and minimized.

Synthetic RNA circuits provide powerful tools to reprogram genetic networks for customized cellular functions. However, the construction of ligand-induced complex synthetic signaling pathways in mammalian cells remains challenging due to the lack of modular and scalable RNA-based components. Here, we report a generalizable strategy to engineer ligand-responsive artificial signaling pathways (ASPs) using programmable RNA circuits. By designing aptamer-embedded circular RNAs as trans-acting triggers coupled with controllable CRISPR functions as outputs, we demonstrate that various small molecules and proteins can be sensed and transduced into manipulation of originally unrelated endogenous genes through amplifiable, logical and multiplexed RNA circuits. Integration of this RNA system into the cellular genetic network endows cells with state/type-specific phenotype responses regulated by endogenous metabolites and proteins. This study establishes a universal RNA platform for engineering ASPs induced by ligands in mammalian cells, with broad potential of cellular signaling and response engineering for diagnostic and therapeutic applications.

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the

Non-canonical CRISPR systems adaptation has led to genome editing through nucleases, and the development of transcriptional and epigenetic regulation, transcriptome editing, and molecular diagnostics has resulted in a diversified set of tools-CRISPR 2.0. In this review, the author summarizes the mechanisms and recent engineering advances of (i) dCas9-based epigenetic effectors, (ii) RNA-targeting Cas13 systems and engineered RNA editors, (iii) DNA base editors and prime editors, and (iv) CRISPR-powered diagnostic platforms and their translational readiness. There is a critical comparison of the various approaches (e.g., RNAi/ASO versus Cas13-based methods; base editing versus prime editing) along with practical translational considerations such as delivery technologies, safety (off-target/edit windows, mosaicism), and regulatory pathways which are evaluated. Three concise case studies refer to map laboratory evidence to clinical or near-clinical outcomes and the ethical and governance discussion is widened to include global access, intellectual property and equity in deployment. Finally, the authors classify technologies according to their level of readiness - diagnostics and some ex-vivo therapeutic approaches are already in or very close to clinical use, chosen in-vivo editing methods are undergoing early trials, and AI-assisted nuclease design is still mostly theoretical but is getting better fast. This comprehensive viewpoint is intended to help researchers and physicians understand which CRISPR tools are most likely to be translated soon and where more validation is required.