Localization of oskar mRNA by agglomeration in ribonucleoprotein granules.

Localization of oskar mRNA to the posterior of the Drosophila oocyte is essential for abdominal patterning and germline development. oskar localization is a multi-step process involving temporally and mechanistically distinct transport modes. Numerous cis-acting elements and trans-acting factors have been identified that mediate earlier motor-dependent transport steps leading to an initial accumulation of oskar at the posterior. Little is known, however, about the requirements for the later localization phase, which depends on cytoplasmic flows and results in the accumulation of large oskar ribonucleoprotein granules, called founder granules, by the end of oogenesis. Using super-resolution microscopy, we show that founder granules are agglomerates of smaller oskar transport particles. In contrast to the earlier kinesin-dependent oskar transport, late-phase localization depends on the sequence as well as on the structure of the spliced oskar localization element (SOLE), but not on the adjacent exon junction complex deposition. Late-phase localization also requires the oskar 3' untranslated region (3' UTR), which targets oskar to founder granules. Together, our results show that 3' UTR-mediated targeting together with SOLE-dependent agglomeration leads to accumulation of oskar in large founder granules at the posterior of the oocyte during late stages of oogenesis. In light of previous work showing that oskar transport particles are solid-like condensates, our findings indicate that founder granules form by a process distinct from that of well-characterized ribonucleoprotein granules like germ granules, P bodies, and stress granules. Additionally, they illustrate how an individual mRNA can be adapted to exploit different localization mechanisms depending on the cellular context.

Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants.

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.