Rapid detection of SARS-CoV-2 RNA in saliva via Cas13.

Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore.

Accelerated RNA detection using tandem CRISPR nucleases.

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.

Accelerated RNA detection using tandem CRISPR nucleases.

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 recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. 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 RNA copies/microliter in 20 minutes.

Rapid, point-of-care molecular diagnostics with Cas13.

Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity.

Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation.

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities.

Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T.

Correction: RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

[This corrects the article DOI: 10.1371/journal.pone.

Reconstitution of the tubular endoplasmic reticulum network with purified components.

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation, but how exactly they mediate network formation and whether they are all required are unclear.

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood.

Cis and trans interactions between atlastin molecules during membrane fusion.

Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes.

A conserved role for atlastin GTPases in regulating lipid droplet size.

Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulated is unknown. Using genetic screens in C. elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size.

Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion.

The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles.

Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes.

The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region.

Screening libraries to identify proteins with desired binding activities using a split-GFP reassembly assay.

Designer protein modules, which bind specifically to a desired target, have numerous potential applications. One approach to creating such proteins is to construct and screen libraries. Here we present a detailed description of using a split-GFP reassembly assay to screen libraries and identify proteins with novel binding properties.

Designed proteins to modulate cellular networks.

A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 10(9) different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework.

Chapter 3. Lymphotactin structural dynamics.

Lymphotactin/XCL1, the defining member of the C class of chemokines, undergoes a conformational change that involves the complete restructuring of all stabilizing interactions. Other chemokines are restricted to a single conformation by a pair of conserved disulfide crosslinks, one of which is absent in lymphotactin. This structural interconversion is entirely reversible, and the two-state equilibrium is sensitive to changes in temperature and ionic strength.