Nucleic-acid detection via isothermal amplification and collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative polymerase chain reaction (qPCR). Here, we report the clinical validation of the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) -the virus that causes COVID-19 (coronavirus disease 2019) -in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per

Thousands of human small and alternative open reading frames (smORFs and alt-ORFs, respectively) have recently been annotated. Many alt-ORFs are co-encoded with canonical proteins in multicistronic configurations, but few of their functions are known. Here, we report the detection of alt-RPL36, a protein co-encoded with human RPL36. Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 increases plasma membrane PI(4,5)P2 levels, upregulates PI3K-AKT-mTOR signaling, and increases cell size. Alt-RPL36 contains four phosphoserine residues, point mutations of which abolish interaction with TMEM24 and, consequently, alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as an upstream regulator of PI3K-AKT-mTOR signaling. More broadly, the RPL36 transcript encodes two sequence-independent polypeptides that co-regulate translation via different molecular mechanisms, expanding our knowledge of multicistronic human gene functions.

While thousands of previously unannotated small and alternative open reading frames (alt-ORFs) have recently been revealed in the human genome, the functions of only a handful are currently known, and no post-translational modifications of their polypeptide products have yet been reported, leaving open the question of their biological significance as a class. Using a proteomic strategy for discovery of unannotated short open reading frames in human cells, wereport the detection of alt-RPL36, a 148-amino acid protein co-encoded with and overlapping human RPL36. Alt-RPL36 interacts with TMEM24, which transports the phosphatidylinositol 4,5bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 in HEK 293T cells increased PI(4,5)P2 levels in the plasma membrane and upregulated the PI3K-AKT-mTOR signaling pathway. Remarkably, we find that four serine residues of alt-RPL36 are phosphorylated, and mutation of these four serines to alanine abolished the interaction with TMEM24 and regulation of PI3K signaling.These results implicate alt-RPL36 as a novel regulator of PI(4,5)P2 synthesis upstream of the PI3K-AKT-mTOR signaling pathway, and the first example of a phosphorylated alt-ORF product.More broadly, both alt-RPL36 and RPL36 regulate protein synthesis and cell growth via different molecular mechanisms -PI3K signaling and ribosome composition, respectively. One human transcript can therefore express two sequence-independent polypeptides from overlapping ORFs that regulate the same processes via distinct mechanisms.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection—such as multiplexed detection for viral variant surveillance—may limit their widespread adoption. Herein, we developed a robust multiplexed CRISPR-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose SARS-CoV-2 infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)—including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)—all while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool—CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance which can be locally manufactured—may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often‐small size and intricate post‐translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue‐specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid‐β peptides, known to be involved in the pathogenesis of Alzheimer's disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.