🧬 PubMed RNA Editing Daily Digest

RNA polymerase III-related disorders represent a clinically diverse spectrum of diseases. While pathogenic variants in the

Regulation of the agonist sensitivity of neurotransmitter receptors is critical for proper functioning of neuronal circuits and is, therefore, conserved across evolutionary time. Mutations that alter agonist sensitivity are often pathological in humans. A brain-expressing nicotinic acetylcholine receptor (nAChR) from the frog Xenopus tropicalis shows ∼20× greater sensitivity to ACh as orthologs from human, chickens, and other frogs prompt us to examine the molecular basis for this extreme sensitivity. We identified a single amino acid substitution in the third transmembrane domain (M3) of the X. tropicalis α4 nAChR subunit, F294 (S in other vertebrate orthologs), that confers the high sensitivity. Surprisingly, we noted variation at this site in sequences deposited in NCBI, suggesting either allelic variation or RNA editing. By sequencing genomic DNA and mRNA (cDNA) from the same individuals from two different colonies of X. tropicalis, we determined that a possible source of this variation is RNA editing. The unedited receptor from X. tropicalis (S294) has a similar ACh sensitivity as those from other vertebrates. Further work must be done to examine possible adaptations of edited receptors and if the frog's brain compensates for an increase in sensitivity since increases in agonist sensitivity lead to pathology in humans.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening technology is redefining the landscape of drug discovery and therapeutic target identification by providing a precise and scalable platform for functional genomics. The development of extensive single-guide RNA (sgRNA) libraries enables high-throughput screening (HTS) that systematically investigates gene-drug interactions across the genome. This powerful approach has found broad applications in identifying drug targets for various diseases, including cancer, infectious diseases, metabolic disorders, and neurodegenerative conditions, playing a crucial role in elucidating drug mechanisms and facilitating drug screening. Despite challenges like off-target effects, data complexity, and ethical or regulatory concerns, ongoing advancements in CRISPR technology and bioinformatics are steadily overcoming these limitations. Additionally, by integrating with organoid models, artificial intelligence (AI), and big data technologies, CRISPR screening expands the scale, intelligence, and automation of drug discovery. This integration boosts data analysis efficiency and offers robust support for uncovering new therapeutic targets and mechanisms. This review outlines the fundamental principles and applications of CRISPR screening technology, delves into specific case studies and technical challenges, and highlights its expanding role in drug discovery and target identification. It also examines the potential for clinical translation and addresses the associated ethical and regulatory considerations.

Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.

Coronaviruses are a family of positive-strand RNA viruses that exhibit highly selective packaging of their genomic RNA (gRNA) into assembled virions, despite the presence of a large excess of subgenomic viral RNA species and host RNA in infected cells. While this high selectivity is critical to evading host innate immune responses, surprisingly, it is not essential for virion assembly. This review focuses on four main topics: (i) coronavirus genome packaging signals (PSs)-how they are found and the function they serve; (ii) the viral components that recognize the PS in order to bring about selective gRNA packaging; (iii) coronavirus nucleocapsid structure and assembly; and (iv) the relationship between nucleocapsid protein phosphorylation and nucleocapsid assembly versus RNA synthesis. Current understanding of these areas has benefited immensely from advances made by recent studies, most of which were performed in response to the emergence of the coronavirus responsible for the COVID-19 pandemic. Throughout this review, emphasis is placed on the counterintuitive distinction between coronavirus selective gRNA packaging and nucleocapsid assembly.