A mechanical-coupling mechanism in OSCA/TMEM63 channel mechanosensitivity.

Mechanosensitive (MS) ion channels are a ubiquitous type of molecular force sensor sensing forces from the surrounding bilayer. The profound structural diversity in these channels suggests that the molecular mechanisms of force sensing follow unique structural blueprints. Here we determine the structures of plant and mammalian OSCA/TMEM63 proteins, allowing us to identify essential elements for mechanotransduction and propose roles for putative bound lipids in OSCA/TMEM63 mechanosensation. Briefly, the central cavity created by the dimer interface couples each subunit and modulates dimeric OSCA/TMEM63 channel mechanosensitivity through the modulating lipids while the cytosolic side of the pore is gated by a plug lipid that prevents the ion permeation. Our results suggest that the gating mechanism of OSCA/TMEM63 channels may combine structural aspects of the 'lipid-gated' mechanism of MscS and TRAAK channels and the calcium-induced gating mechanism of the TMEM16 family, which may provide insights into the structural rearrangements of TMEM16/TMC superfamilies.

Mechanism underlying delayed rectifying in human voltage-mediated activation Eag2 channel.

The transmembrane voltage gradient is a general physico-chemical cue that regulates diverse biological function through voltage-gated ion channels. How voltage sensing mediates ion flows remains unknown at the molecular level. Here, we report six conformations of the human Eag2 (hEag2) ranging from closed, pre-open, open, and pore dilation but non-conducting states captured by cryo-electron microscopy (cryo-EM). These multiple states illuminate dynamics of the selectivity filter and ion permeation pathway with delayed rectifier properties and Cole-Moore effect at the atomic level. Mechanistically, a short S4-S5 linker is coupled with the constrict sites to mediate voltage transducing in a non-domain-swapped configuration, resulting transitions for constrict sites of F464 and Q472 from gating to open state stabilizing for voltage energy transduction. Meanwhile, an additional potassium ion occupied at positions S6 confers the delayed rectifier property and Cole-Moore effects. These results provide insight into voltage transducing and potassium current across membrane, and shed light on the long-sought Cole-Moore effects.

Embigin facilitates monocarboxylate transporter 1 localization to the plasma membrane and transition to a decoupling state.

Cell-surface ancillary glycoproteins basigin or embigin form heterodimeric complexes with proton-coupled monocarboxylate transporters (MCTs), facilitating the membrane trafficking of MCTs and regulating their transport activities. Here, we determine the cryoelectron microscopy (cryo-EM) structure of the human MCT1-embigin complex and observe that embigin forms extensive interactions with MCT1 to facilitate its localization to the plasma membrane. In addition, the formation of the heterodimer effectively blocks MCT1 from forming a homodimer through a steric hindrance effect, releasing the coupling between two signature motifs and driving a significant conformation change in transmembrane helix 5 (TM5) of MCTs. Consequently, the substrate-binding pocket alternates between states of homodimeric coupling and heterodimeric decoupling states and exhibits differences in substrate-binding affinity, supporting the hypothesis that the substrate-induced motion originating in one subunit of the MCT dimer could be transmitted to the adjacent subunit to alter its substrate-binding affinity.

Phase separating cell fate.

How master regulators such as Oct4 can shape 3D genome architecture during cell reprogramming remains poorly understood. In this issue of Cell Stem Cell, Wang et al. (2021) demonstrate that Oct4 undergoes phase separation to reconfigure TAD architecture for cell fate control.

A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 µL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.

Cascade CRISPR/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity.

Existing CRISPR/cas-based biosensors usually improve sensitivity by target amplification, which is time-consuming and susceptible to impurities in complex biofluid. Herein, this is the first time a cascade CRISPR/cas (casCRISPR) system has been developed, which can provide a detection limit of 1.33 fM (∼1000 times lower than direct Cas13a-based miRNA detection) and single-base resolution for miR-17 detection without resorting to target amplification. casCRISPR can also be applied to detect miRNA in complicated cell extracts and serum samples. Overall, casCRISPR will provide a heuristic idea for CRISPR/cas based biosensing, and could be a promising tool for miRNA diagnostics.

Delivery of Cas13a/crRNA by self-degradable black phosphorus nanosheets to specifically inhibit Mcl-1 for breast cancer therapy.

Mcl-1 amplification has been observed in breast cancer and demonstrated as a key determinant of breast cancer cell survival. However, the clinical use of available effective Mcl-1-specific inhibitors for breast cancer treatment remains a challenge. An RNA-guided CRISPR/Cas13a system targeting RNAs can be used to specifically knock down mRNA expression in mammalian cells. The goal of this work is to develop a self-degradable nanoplatform based on polylysine (PLL)-functionalized black phosphorus (PBP) for the delivery of Cas13a/crRNA complexes to specifically inhibit Mcl-1 at transcriptional level for breast cancer therapy. The constructed Cas13a/crRNA complex is delivered into the cytoplasm by PBP via endocytosis, followed by endosomal escape based on the biodegradation of PBP, and this efficiently knocks down the specific gene at transcriptional level up to an efficiency of 58.64%. Through designing CRISPR RNA crMcl-1, Mcl-1 can be specifically knocked down at transcriptional level in breast cancer cells, resulting in the down-regulation of the expression of Mcl-1 protein and inhibition of the cell activity. Notably, PBP/Cas13a/crMcl-1 shows an excellent tumor suppression efficacy up to 65.16% after intratumoral injection. Therefore, biodegradable PBP is an ideal nanoplatform for the delivery of CRISPR/Cas13a, which could provide a potential strategy for gene therapy.

CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection.

MicroRNAs (miRNAs) have been widely investigated as potential biomarkers for early clinical diagnosis of cancer. Developing an miRNA detection platform with high specificity, sensitivity, and exploitability is always necessary. Electrochemiluminescence (ECL) is an electrogenerated chemiluminescence technology that greatly decreases background noise and improves detection sensitivity. The development of a paper-based ECL biosensor further makes ECL suitable for point-of-care detection. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a as high-fidelity, efficient, and programmable CRISPR RNA (crRNA) guided RNase has brought a next-generation biosensing technology. However, existing CRISPR/Cas13a based detection often faces a trade-off between sensitivity and specificity. In this research, a CRISPR/Cas13a powered portable ECL chip (PECL-CRISPR) is constructed. Wherein target miRNA activates Cas13a to cleave a well-designed preprimer, and triggers the subsequent exponential amplification and ECL detection. Under optimized conditions, a limit-of-detection of 1 × 10-15 m for miR-17 is achieved. Through rationally designing the crRNA, the platform can provide single nucleotide resolution to dramatically distinguish miRNA target from its highly homologous family members. Moreover, the introduction of "light-switch" molecule [Ru(phen)2dppz]2+ allows the platform to avoid tedious electrode modification and washing processes, thereby simplifying the experimental procedure and lower testing cost. Analysis results of miRNA from tumor cells also demonstrate the PECL-CRISPR platform holds a promising potential for molecular diagnosis.

Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.

The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called 'APC-Cas') that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 105 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.

High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity.

MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. It has been proved that the aberrant expression of miRNAs is related to disease and miRNAs can serve as potential biomarkers for early tumor diagnosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a is a recently discovered CRISPR-RNA (crRNA) guided RNA manipulation tool. The recognition of target RNA can morphologically activate the robust nonspecific trans ribonuclease activity of Cas13a. This unique property makes Cas13a ideal for nucleic acid detection. Herein, we first exploited CRISPR/LbuCas13a to directly detect miRNAs with high specificity and simplicity. A limit of detection (LOD) as low as 4.5 amol was achieved by this one-step assay within 30 min, and the dynamic range spanned 4 orders of magnitude from 10 amol to 100 fmol. More importantly, single nucleotide variation, even at the end of target miRNA, can be discriminated by rationally programmed crRNA. In addition, the practical application ability of this Cas13a/crRNA-based signal amplification strategy was demonstrated by miRNA quantification in complex biological samples (total small RNA). With excellent reliability, sensitivity, and simple to implement features, this method promises a great potential for early diagnosis of miRNA-related disease. Moreover, the systematic analysis of the crRNA design could provide guidance to further develop Cas13a-based molecular diagnoses.