🧬 PubMed RNA Editing Daily Digest

Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.

RNA-based biosensors have emerged as essential tools in synthetic biology and diagnostics, enabling precise and programmable responses to diverse RNA inputs. However, the time to design, produce, and screen high-performance RNA sensors remains a critical challenge. The fundamental rules governing RNA-RNA interactions-specifically the structure-function relationships that determine sensor performance-remain poorly understood. Here, we present a method enabling versatile in-silico RNA-targeting analysis (VISTA), a machine learning-guided framework for the rapid design of RNA sensors. VISTA integrates biophysical modeling of both sensor and target RNAs with a partial least squares discriminant analysis machine learning framework. Using high-throughput experimental measurements with sequence-structure feature extraction to train predictive models, we capture the key determinants of RNA sensor performance. By using toehold switches as a model RNA sensor, we find that Toehold-VISTA successfully designs RNA sensors with improved performance against SARS-CoV-2 RNA. These findings establish a broadly applicable, target-aware design strategy for accelerating RNA sensor engineering across biotechnology and diagnostic applications.

Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.

Ischemic stroke (IS) is one of the most common neurological diseases worldwide and is caused by the blockage of cerebral blood vessels, leading to reduced blood flow and neuronal damage. Given the limitations of existing treatments, CRISPR gene-editing technology has emerged as a promising strategy to precisely target the molecular pathways underlying IS pathophysiology. By enabling intervention in genes regulating inflammation, apoptosis, and repair, CRISPR enables more precise and effective therapies. Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells. Studies have shown that the use of CRISPR-Cas9 to modulate key pathogenic pathways, including those governing inflammation, oxidative stress, and cell death, can prevent neuronal damage and improve neurological function. Additionally, targeting ncRNAs and RNA methylation with CRISPR-based systems plays a role in regulating oxidative stress and stress granule formation. The use of CRISPR to modulate cell communication and organelle transfer and correct mitochondrial mutations has also been considered a neuroprotective mechanism. Despite persistent challenges in targeted and safe delivery, substantial preclinical advances, primarily in rodent models, underscore the potential for CRISPR-based therapies to transform future stroke treatment. These findings suggest that CRISPR-based strategies could evolve into precision neurotherapeutics that address root molecular pathologies, potentially complementing or surpassing current stroke interventions.

Chloroplast gene expression and regulation are essential for plant growth, development, and environmental adaptation, in which post-transcriptional RNA editing and splicing processing play an important role. This study demonstrates that OsDYW2, a rice DYW domain-containing protein, is crucial for both chloroplast RNA editing and splicing, particularly under high temperature. Mutants lacking OsDYW2 exhibit a high-temperature-sensitive albino lethal phenotype, characterized by defective chloroplast biogenesis and suppressed expression of chloroplast development and photosynthesis-associated genes. The loss of OsDYW2 function impairs the RNA editing of matK-1258, ndhA-473, ndhA-563, ndhA-1070, ndhB-586, ndhB-611, ndhB-737, ndhB-830, ndhD-878, ndhG-5-UTR-10, ndhG-347, rpl2-2, rpoC2-4106, and rps14-80 sites as well as the RNA splicing of atpF, ndhA, ndhB, petB, rpl2, rps12, and rps16 under high-temperature conditions. Mechanistically, OsDYW2 interacts with core RNA processing factors, including several rice multiple organellar RNA editing factor (OsMORF) proteins, to form a temperature-sensitive complex. These interactions are modulated by high temperature, suggesting a direct link between high-temperature response and chloroplast RNA processing. Our findings demonstrate that OsDYW2 is essential for coordinating chloroplast post-transcriptional gene expression regulation with the high-temperature stress response in rice, providing new insights into the molecular mechanisms underlying chloroplast adaptation to environmental challenges.