🧬 PubMed RNA Editing Daily Digest

IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB's regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide-target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide-target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a 'car pedal' motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a widespread modification in metazoans. Cumulative evidence has revealed the altered A-to-I editing profiles in cancers, but the underlying mechanism remains unclear. Here, we discover the well-known histone lysine methyltransferase enhancer of zeste homologue 2 (EZH2) as an unexplored ADAR interactor and editing regulator in prostate cancer (PCa). Through competing with interleukin enhancer binding factor 2 (ILF2) for ADAR1 binding, EZH2 reshapes the substrate selectivity of ADAR1 and thus exhibits a bidirectional role in editing regulation. Moreover, EZH2 depletion induces the translational repression of transportin-1 (TRN1), which further results in the accumulation of cytoplasmic ADAR1p110 isoform to protect many oncogenic transcripts from degradation. Consistently, depletion of ADAR1 dramatically enhances the sensitivity of cancer cells and tumors to EZH2 selective degraders. Collectively, our study sheds new light on a link between two layers of epigenetic regulations at histone modification and RNA editing levels, demonstrates a previously uncharacterized role of EZH2 in RNA editing and mRNA stability independently of its lysine methyltransferase activity, and reveals the significance of EZH2-ADAR1 cascade in governing RNA editing and mRNA stability, which may provide additional perspectives for the advancement of EZH2-targeting cancer therapies.

Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the "gold standard" quantitative reverse transcription-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA), during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared the results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A positivity, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces. Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal coronaviruses, respiratory syncytial virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.

Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR-dCas9-based tools offer this potential, yet β-cells are notoriously difficult to transfect, and efficient, non-viral delivery methods are lacking. Here, we developed nanopore-mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β-cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel-associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin-1 gene (Ins1) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult-to-transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR-dCas9-based epigenetic editors in pancreatic β-cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.

Genetically engineering human pluripotent stem cell (hPSC)-derived islets is a promising strategy for improving transplantation for diabetes cell therapy; however, genetic perturbations that modulate transplantation outcomes have yet to be systematically explored. To identify potential targets, we performed an unbiased whole-genome CRISPR-activation screen in transplanted stem cell-derived islets (SC-islets). Specifically, we created a stem cell line with CRISPR-activation components (HUES8-VPR) and then transduced these stem cells with a lentiviral guide RNA library targeting the whole human genome. Following transduction, the stem cells were differentiated into SC-islets, which were subsequently transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) immunodeficient mice. After transplantation, SC-islets were extracted for next-generation sequencing. The screen identified multiple candidates, including the Fc alpha/mu receptor (FCAMR). In vitro characterization revealed that FCAMR overexpression did not negatively affect SC-islet function or transcriptomic identity. Mice subcutaneously transplanted with SC-islets overexpressing FCAMR had reduced blood glucose levels and increased C-peptide compared to controls. Additionally, mice receiving FCAMR-modified grafts into the kidney capsule or hindleg muscle maintained a higher body weight compared to controls in a diabetic setting. In conclusion, this study demonstrats improved glucose regulation at a subcutaneous transplant site. In addition, we show that FCAMR SC-islets could play a role in systemic metabolism when transplanted into the kidney capsule or hindleg muscle. Overall, our study establishes a functional screening approach to identify gene candidates to improve SC-islet transplantation.