ABSTRACT
The cellular process by which the protein ubiquitin (Ub) is covalently attached to a protein substrate involves Ub activating (E1s) and conjugating enzymes (E2s) that work together with a large variety of E3 ligases that impart substrate specificity. The largest family of E3s is the Cullin-RING ligase (CRL) family which utilizes a wide variety of substrate receptors, adapter proteins, and cooperating ligases. Cryo-electron microscopy (cryoEM) has revealed a wide variety of structures which suggest how Ub transfer occurs. Hydrogen deuterium exchange mass spectrometry (HDXMS) has revealed the role of dynamics and expanded our knowledge of how covalent NEDD8 modification (neddylation) activates the CRLs, particularly by facilitating cooperation with additional RING-between-RING ligases to transfer Ub.
ABSTRACT
The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/µL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , RNA, Viral , RuminococcusABSTRACT
Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.
