Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C t ) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.
ObjectivePrevious studies have shown that differential DNA methylation is associated with SLE susceptibility. How DNA methylation affects the clinical heterogeneity of SLE has not been fully defined. We conducted this study to identify differentially methylated CpG sites associated with nephritis among women with SLE.MethodsThe methylation status of 428 229 CpG sites across the genome was characterised for peripheral blood cells from 322 women of European descent with SLE, 80 of whom had lupus nephritis, using the Illumina HumanMethylation450 BeadChip. Multivariable linear regression adjusting for population substructure and leucocyte cell proportions was used to identify differentially methylated sites associated with lupus nephritis. The influence of genetic variation on methylation status was investigated using data from a genome-wide association study of SLE. Pathway analyses were used to identify biological processes associated with lupus nephritis.ResultsWe identified differential methylation of 19 sites in 18 genomic regions that was associated with nephritis among patients with SLE (false discovery rate q<0.05). Associations for four sites in HIF3A, IFI44 and PRR4 were replicated when examining methylation data derived from CD4+ T cells collected from an independent set of patients with SLE. These associations were not driven by genetic variation within or around the genomic regions. In addition, genes associated with lupus nephritis in a prior genome-wide association study were not differentially methylated in this epigenome-wide study. Pathway analysis indicated that biological processes involving type 1 interferon responses and the development of the immune system were associated with nephritis in patients with SLE.ConclusionsDifferential methylation of genes regulating the response to tissue hypoxia and interferon-mediated signalling is associated with lupus nephritis among women with SLE. These findings have not been identified in genetic studies of lupus nephritis, suggesting that epigenome-wide association studies can help identify the genomic differences that underlie the clinical heterogeneity of SLE.
ObjectiveEpigenetic modifications have previously been associated with rheumatoid arthritis (RA). In this study, we aimed to determine whether differential DNA methylation in peripheral blood cell subpopulations is associated with any of 4 clinical outcomes among RA patients.MethodsPeripheral blood samples were obtained from 63 patients in the University of California, San Francisco RA cohort (all satisfied the American College of Rheumatology classification criteria; 57 were seropositive for rheumatoid factor and/or anti‐cyclic citrullinated protein). Fluorescence‐activated cell sorting was used to separate the cells into 4 immune cell subpopulations (CD14+ monocytes, CD19+ B cells, CD4+ naive T cells, and CD4+ memory T cells) per individual, and 229 epigenome‐wide DNA methylation profiles were generated using Illumina HumanMethylation450 BeadChips. Differentially methylated positions and regions associated with the Clinical Disease Activity Index score, erosive disease, RA Articular Damage score, Sharp score, medication at time of blood draw, smoking status, and disease duration were identified using robust regression models and empirical Bayes variance estimators.ResultsDifferential methylation of CpG sites associated with clinical outcomes was observed in all 4 cell types. Hypomethylated regions in the CYP2E1 and DUSP22 gene promoters were associated with active and erosive disease, respectively. Pathway analyses suggested that the biologic mechanisms underlying each clinical outcome are cell type–specific. Evidence of independent effects on DNA methylation from smoking, medication use, and disease duration were also identified.ConclusionMethylation signatures specific to RA clinical outcomes may have utility as biomarkers or predictors of exposure, disease progression, and disease severity.
To combat disease outbreaks such as the COVID-19 pandemic, flexible diagnostics for rapid viral detection are greatly needed. We report a nucleic acid test that integrates distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We paired this workflow, termed Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER), with extraction-free sample lysis using shelf-stable reagents that are widely available at low cost. DISCoVER has been validated on saliva samples to incentivize frequent testing for more widespread community surveillance and robustly detected attomolar levels of SARS-CoV-2 within 30 minutes, while avoiding false positives in virus-negative saliva. Furthermore, DISCoVER is compatible with multiplexed CRISPR probes to enable simultaneous detection of a human gene control or alternative pathogens.
The phagocyte oxidative burst, mediated by Nox2 NADPH oxidase-derived reactive oxygen species, confers host defense against a broad spectrum of bacterial and fungal pathogens. Loss-of-function mutations that impair function of the Nox2 complex result in a life-threatening immunodeficiency, and genetic variants of Nox2 subunits have been implicated in pathogenesis of inflammatory bowel disease (IBD). Thus, alterations in the oxidative burst can profoundly impact host defense, yet little is known about regulatory mechanisms that fine-tune this response. Here we report the discovery of regulatory nodes controlling oxidative burst by functional screening of genes within loci linked to human inflammatory disease. Implementing a multi-omics approach, we define transcriptional, metabolic and ubiquitin-cycling nodes controlled by Rbpj, Pfkl and Rnf145, respectively. Furthermore, we implicate Rnf145 in proteostasis of the Nox2 complex by endoplasmic reticulum-associated degradation. Consequently, ablation of Rnf145 in murine macrophages enhances bacterial clearance, and rescues the oxidative burst defects associated with Ncf4 haploinsufficiency.
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