🧬 PubMed RNA Editing Daily Digest

While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.

Reduced-size CRISPR systems have become a possible remedy to the delivery and size constraints of the traditional SpCas9 (~ 1368 Å). Recently described small nucleases, including Cas12f (400-700 Å) or CasX (~ 980 Å), along with designed mini-Cas9 versions, can efficiently be used in vivo to edit cells as well as to perform point-of-care diagnostics because of their lower molecular weight and less complex structures. This review will sum up progress in compact Cas protein engineering, guide RNA optimization, and delivery vector miniaturization, and point to their influence in therapeutic gene editing and portable diagnostic platforms. We additionally cover the contemporary issues of interest, such as off-target activity, delivery barriers and regulatory requirements, and future opportunities provided through AI-assisted protein design and synthetic biology. The miniaturized CRISPR technology is bound to substantially transform the translational arena of gene editing and world diagnostics.

RNA editing is a post-transcriptional modification that expands transcriptomic and proteomic diversity. Advances in high-throughput sequencing across a broad range of biological and pathological contexts have enabled systematic identification of editing events driven by two major RNA deaminase families: ADAR and APOBEC, which catalyze adenosine-to-inosine (A-to-I) and cytidine-to-uridine (C-to-U) substitution, respectively. Genome-wide profiling of RNA editing has uncovered a substantial number of differentially edited loci in various conditions, implicating the post-transcriptional events in physiological and pathological regulation. Aberrant RNA editing alters the functional information of coding and noncoding transcripts, perturbing protein activity, RNA stability and other gene expression programs, which contributes to immune imbalance, viral infection, neurological impairment, metabolic disorders and tumorigenesis. The two codes of A-to-I and C-to-U RNA editing harbor common potential for single base conversion with varied expression of responsible enzymes across many physiological and pathological conditions. Here we provide a comprehensive and parallel overview on ADAR-mediated A-to-I and APOBEC-mediated C-to-U editing, with emphasis on their molecular mechanisms, physiological roles and pathological dysregulation in human health and disease.

Okazaki fragment maturation requires efficient removal of RNA primers to form a continuous lagging strand, yet how mismatched primers introduced by error-prone primase are corrected remains unresolved. Here, we show that physiological levels of reactive oxygen species (ROS) initiate a redox-dependent mechanism that drives ADAR1-mediated adenosine-to-inosine (A-to-I) editing. Oxidation triggers ADAR1 dimerization at replication forks, enhancing RNA editing of mismatched primers-particularly those caused by ATP misincorporation on d(T+C)-rich centromeric DNA. This A-to-I editing step facilitates more efficient RNA primer degradation by RNase H2, thereby ensuring proper Okazaki fragment maturation. Disruption of ADAR1 oxidation results in increased unligated Okazaki fragments, single-stranded gaps and double-strand breaks, most prominently at centromeres. These findings reveal a role for ROS in safeguarding lagging-strand synthesis by coupling ADAR1 oxidation-induced A-to-I RNA editing to replication fork stability.

Mitochondrial genomes play essential roles in plant energy metabolism and evolution, yet their structural complexity and diversity in plants remain poorly understood. This study aims to address the question by analyzing four newly assembled Mentha mitochondrial genomes (M. longifolia, M. suaveolens, M. pulegium, and M. requienii), which serve as valuable genomic resources for phylogenetic and evolutionary studies. Comparative analyses revealed structural diversity, codon usage bias, extensive RNA editing, and abundant repetitive sequences driving genomic rearrangements in the four mitochondrial genomes. Chloroplast-derived DNA fragments were dynamically integrated into the four Mentha mitochondrial genomes, highlighting ongoing interorganellar DNA transfer between plastids and mitochondria. Phylogenetic reconstructions based on mitochondrial, nuclear, and chloroplast genomes exhibit considerable discordance, reflecting complex evolutionary processes such as hybridization, introgression, and allopolyploidization within the genus. In conclusion, the structural diversity, codon usage bias, and ongoing interorganellar DNA transfer observed in Mentha mitochondrial genomes underscore their dynamic evolutionary nature. The discordance among mitochondrial, plastid, and nuclear phylogenies reflects complex evolutionary processes (possibly hybridization and allo-polyplodization) of Mentha species. These findings enhance the understanding of the mechanisms underlying the complexity and diversity of Mentha species and provide broader insights into the evolution of plant mitochondrial genomes.