The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.
Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.
The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR’s sensor domain and by front-to-front interfaces between PKR’s kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.
Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.
IMPORTANCE The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. OBJECTIVE To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPRbased test developed for accessible and large-scale viral screening. DESIGN, SETTING, AND PARTICIPANTS For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self
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