🧬 PubMed RNA Editing Daily Digest

Programmable A-to-I RNA editing using endogenous ADAR enzymes is emerging as a therapeutic strategy, but editability remains difficult to predict because ADAR recognition depends on double-stranded RNA geometry and stability rather than sequence alone. We present A dar E dit , a structure-explicit graph-attention framework that represents each dsRNA substrate as a nucleotide graph with backbone and base-pair edges and augments this representation with typed interactions and a motif-sensitive sequence branch. We trained and evaluated the model on high-confidence inverted

Modified mRNA technology has transformed vaccine development by enabling rapid and precise antigen production. The incorporation of adjuvants may further enhance innate immune activation, thereby improving the efficacy of mRNA vaccines. However, inappropriate vaccine kinetics leading to excessive activation of the innate immune system can inhibit the mTORC1 pathway and impair antigen mRNA translation, ultimately limiting vaccine potency. Herein, we present the development of a time-ordered-expression mRNA (TOE mRNA) based on ADAR-mediated A-to-I base editing. Upon cellular entry, the TOE mRNA initiates immediate antigen translation, while the adjuvant encoded within the same mRNA is translated approximately 12 hours post-antigen expression. This delayed adjuvant expression ensures sustained activation of the mTORC1 pathway and robust antigen expression, effectively overcoming the limitations imposed by suboptimal vaccine kinetics. We demonstrate that vaccines utilizing TOE mRNA encoding a tumor neoantigen (for normal translation) and an IL-12 adjuvant (with delayed translation) elicit significantly enhanced antitumor immune responses. TOE mRNA technology represents a promising platform for advancing next-generation mRNA vaccines with improved efficacy.

CRISPR diagnostics have emerged as powerful tools for detecting infectious diseases, with the RNA endonuclease Cas13a enabling sensitive and specific, amplification-free RNA detection through collateral

CRISPR activation and interference systems (CRISPRa/i) are widely used for programmable transcriptional control. Although these technologies are capable of highly specific single-gene activity, some applications of transcriptional network reprogramming require broad, genome-wide effects. Here, we identify a CRISPRa gRNA that robustly reprograms astrocyte transcriptional state. Unexpectedly, this activity arises from extensive off-target binding that induces expression changes in thousands of genes, unlike neighboring gRNAs targeting the same intended on-target site. We leverage this promiscuous gRNA to dissect determinants of gRNA-driven off-target dCas9 binding in the context of transcriptional reprogramming. Using ChIP-seq, high-throughput protein-binding microarrays, and gRNA-variant library screening in cells, we demonstrate that PAM-proximal bases are primary determinants of genomic binding, mismatch tolerance is both gRNA- and base-specific, and targeted mutations within the PAM-proximal region can tune gRNA specificity. We further demonstrate that CRISPRa-driven phenotypes can reflect combined contributions from widespread off-target activity and dose-dependent on-target effects. These findings highlight the potentially widespread impacts of CRISPRa off-target activity, underscore the need to account for cryptic effects when selecting and evaluating gRNAs for programming cell phenotypes, and demonstrate that multi-site binding by CRISPRa systems can be exploited as a feature for network-level perturbations in cell reprogramming.

Alpha-1 antitrypsin deficiency (AATD) is a rare, underdiagnosed genetic disorder characterized by deficient or dysfunctional alpha-1 antitrypsin (AAT), leading to unopposed neutrophil protease activity. This can often lead to progressive lung and liver damage, and in rare cases panniculitis, which can be potentially lethal. Current diagnostic strategies rely on a stepwise approach beginning with serum AAT measurement, followed by phenotyping and genotyping to confirm pathogenic variants. Despite recommendations from major respiratory societies to test all patients with chronic obstructive pulmonary disease (COPD), unexplained liver disease, panniculitis, and vasculitis, delayed recognition persists because of clinical overlap with asthma, COPD, and alcohol- and non-alcohol-related liver disease. Management emphasizes lifestyle modification, avoidance of risk factors, and pharmacological support. Currently, intravenous augmentation therapy is the only disease-modifying option approved for pulmonary disease. Augmentation is expensive and variably available, or reimbursed worldwide. Surgical and interventional approaches, including lung volume reduction techniques and transplantation, provide options for advanced disease, although outcomes vary. Emerging therapies, such as inhaled AAT formulations, recombinant fusion proteins, RNA-editing platforms, and DNA editing using CRISPR-based strategies, aim to correct the protease-antiprotease imbalance and restore endogenous AAT production. The therapeutic landscape is rapidly evolving, but significant challenges remain in improving early detection, broadening access to effective treatments, and optimizing individualized care. Future advances will likely depend on integrating newer therapies with early intervention strategies to preserve organ function and improve long-term prognosis.