🧬 PubMed RNA Editing Daily Digest

Lateral flow assay (LFA) is a versatile platform for the rapid detection of various pathogens, pollutants, and toxins at point of care (POC). However, a traditional lateral flow assay strip is still not sensitive enough for early detection of disease, and previous works to enhance the sensitivity of LFA systems have suffered from additional signal amplification chemistry, tedious operation procedure, or expensive signal amplification instruments. In this study, we introduced a digital counting strategy for signal measurement to improve the detection sensitivity of quantum dot nanobead-based lateral flow immunoassays, offering an alternative to traditional fluorescence intensity-based quantification. The ultrasensitive single-molecule POC detection system is composed of an ultrabright fluorescent quantum dot nanobead-based lateral flow assay strip and a portable fluorescent microscopy reader for numerating single immunocomplexes on a test line. As a proof-of-concept, we performed digital LFA to detect interleukin-6 in 1% BSA with LoD as low as 0.02 pg/mL, which is ∼100 times more sensitive than analog LFA. Moreover, an amplification-free assay for HIV RNA was also demonstrated by combining CRISPR-Cas13 reaction and digital LFA with a sensitivity of as low as 89 copies/mL. A prototype portable and affordable fluorescent microscopy reader was designed and demonstrated its ability to be used for on-site testing. All in all, the developed digital LFA system not only has single-molecule level sensitivity but also satisfies the rapidity, easy operation, portability, and low-cost requirements for POC testing.

Adenosine deaminase acting on RNA 1 (ADAR1) regulates mRNA fate and function through adenosine-to-inosine (A-to-I) RNA editing and RNA-binding activities. While its role in innate immunity is established, the broader regulatory functions of ADAR1 in macrophages remain poorly defined. Here, we systematically profiled ADAR1 expression across human immune cells and identified marked enrichment in macrophages, driven by selective usage of an alternative transcription start site during monocyte-to-macrophage differentiation. ADAR1 binds, edits, and modulates key macrophage targets involved in efferocytosis, endocytosis, lysosomal processing, lipid metabolism, and proliferation in an isoform-specific manner. We further demonstrate that ADAR1 levels and activity are dynamically regulated in adipose tissue and liver during the progression of metabolic disease. Linked to this, macrophage-specific ablation of ADAR1 co-cultured in organotypic 3D primary human liver spheroids and exposed to metabolic stress resulted in an exacerbated lipid accumulation phenotype. Finally, we identify a lipid-associated macrophage-specific upregulation of ADAR1 in adipose tissue following weight loss interventions, mechanistically driven by free fatty acids. These findings uncover a previously unrecognized role for ADAR1 in lipid-buffering, scavenging, and proliferative macrophage functions, extending its biological relevance beyond canonical interferon-mediated immunity and establishing ADAR1 as a key regulator of macrophage adaptation in metabolic disease.

Multiplex genome editing using the CRISPR/Cas9 system allows simultaneous modifications at several genomic sites, offering great potential for crop improvement. Among various approaches, the polycistronic tRNA-gRNA (PTG) system is widely adopted due to its use of the host's native tRNA processing machinery, enabling the generation of multiple sgRNAs from a single transcript without the need for expressing any foreign RNA processing enzymes or ribozymes. However, designing the complete set of primers suitable for performing in vitro PTG assembly is complex and needs expertise, as a single mistake can lead to complete failure of the assembly process or subsequent editing. To overcome this challenge, we developed CRISP-PTG-Assembler Ver. 1.0, a user-friendly tool that takes only (i) 20-nucleotide sgRNA spacers and (ii) 4-nucleotide joiners as inputs; and produces colour-coded outputs in forms of (i) Primer-set required for complete PTG assembly, (ii) Primary PCR Amplicons, (iii) Overlap-Extension PCR Amplicons and (iv) Expected PTG assembly, for easy interpretation and construct making. Our novel assembly approach provides flexibility in sticky-end choice during golden gate ligation and ensures the fidelity of component sgRNAs in the PTG assembly by buffering against ligation errors (~ 1.5-40%) that may occur during the Golden Gate assembly process, thereby safeguarding the functionality of the in vivo-generated individual sgRNA molecules. We validated its effectiveness by editing two loci of the matrix metalloproteinase 1 gene in rice and demonstrated its applicability across various plant systems. With an intuitive interface and robust features, CRISP-PTG-Assembler empowers researchers of all levels to effectively implement PTG-based multiplex genome editing in plants.

We established a step-by-step approach for generating a single-nucleotide mutation in the promoter region of an immune regulatory gene in human monocyte THP-1 cells by employing a plasmid-based CRISPR-Cas9 system delivered via transfection with a homology-directed repair template DNA (HDR). Key steps include designing a single-guide RNA (sgRNA), cloning it into a CRISPR plasmid encoding the Cas9 protein, transfection of the plasmid constructs along with single-stranded oligonucleotide repair template (ssODNs) into THP-1 cells, followed by selection and validation. This approach provides a precise and relevant model to investigate the role of single polymorphisms in the regulation of inflammatory gene expression in human monocytes. In addition to the rs1024611 single-nucleotide polymorphism (SNP), this CRISPR/Cas9-based strategy is broadly applicable to functional studies of noncoding and coding variants in innate immune genes. Key features • Precise genome editing: CRISPR-Cas9-mediated editing of the rs1024611 SNP in the endogenous

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