Engineering Anti-CRISPR Proteins to Create CRISPR-Cas Protein Switches for Activatable Genome Editing and Viral Protease Detection.

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.

Buried Defect Detection Method for a Blowout Preventer Seal Ring Groove Based on an Ultrasonic Phased Array.

This study aims to investigate an accurate detection method to detect defects in the gasket ring groove of the blowout preventer (BOP) using the ultrasonic phased array technology. Traditionally, it is difficult to accurately determine the type and size of defects in the gasket ring groove due to the complexity of the BOP configuration and the interference between the defect echo and the structural echo when using the ultrasonic phased array detection technology. In this study, firstly, the appropriate detection process parameters are determined by using simulation software for simulating and analyzing the defects of different sizes and types in the gasket ring groove of a BOP. Thereafter, according to the detection process parameters determined by the simulation analysis, we carry out a corresponding actual detection test. Simulation analysis and detection test results show that the relative amplitude of the test results and the simulation results differ within 1 dB, and the simulation results have a guiding role for the actual detection. The defect echo and structure echo can be clearly distinguished by selecting appropriate detection process parameters, such as probe frequency 5 MHz, array elements 36, and probe aperture 16 mm. The research results can provide theoretical reference for the detection of blowout preventer.

The IPGA1-ANGUSTIFOLIA module regulates microtubule organisation and pavement cell shape in Arabidopsis.

Plant cells continuously experience mechanical stress resulting from the cell wall that bears internal turgor pressure. Cortical microtubules align with the predicted maximal tensile stress direction to guide cellulose biosynthesis and therefore results in cell wall reinforcement. We have previously identified Increased Petal Growth Anisotropy (IPGA1) as a putative microtubule-associated protein in Arabidopsis, but the function of IPGA1 remains unclear. Here, using the Arabidopsis cotyledon pavement cell as a model, we demonstrated that IPGA1 forms protein granules and interacts with ANGUSTIFOLIA (AN) to cooperatively regulate microtubule organisation in response to stress. Application of mechanical perturbations, such as cell ablation, led to microtubule reorganisation into aligned arrays in wild-type cells. This microtubule response to stress was enhanced in the IPGA1 loss-of-function mutant. Mechanical perturbations promoted the formation of IPGA1 granules on microtubules. We further showed that IPGA1 physically interacted with AN both in vitro and on microtubules. The ipga1 mutant alleles exhibited reduced interdigitated growth of pavement cells, with smooth shape. IPGA1 and AN had a genetic interaction in regulating pavement cell shape. Furthermore, IPGA1 genetically and physically interacted with the microtubule-severing enzyme KATANIN. We propose that the IPGA1-AN module regulates microtubule organisation and pavement cell shape.

A switchable Cas12a enabling CRISPR-based direct histone deacetylase activity detection.

The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by anti-CRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics.

An all-in-one telomerase assay based on CRISPR-Cas12a trans-cleavage while telomere synthesis.

As one of the crucial factors associated with human life span and cancer progression, telomerase is regarded as an emerging biomarker for cancer diagnosis. Therefore, a facile, rapid and sensitive approach for telomerase activity detection with point-of-care (POC) diagnosis potential is in great demands. Herein, an all-in-one telomerase activity detection assay was established based on the telomere synthesis activated CRISPR-Cas12a system. A telomerase extension reaction generated telomere repeats sequences (TTAGGG)n, which was recognized by a customized CRISPR-guided RNA (crRNA) simultaneously, and finally activated a typical trans-cleavage based CRISPR-Cas12a detection assay. With the inherent sensitivity of CRISPR-Cas12a, this approach achieved a great linear regression ranging from 100 to 2000 HeLa cells and a limitation of detection down to 26 HeLa cells. Moreover, by using the proposed method, telomerase can be detected in one pot under isothermal condition (37 °C) by a simple and fast workflow (one step within 1 h). Due to its excellent performance, this all-in-one method shows great potential in POC detection of the telomerase activity.

Reactive oxygen species mediate conical cell shaping in Arabidopsis thaliana petals.

Plants have evolved diverse cell types with distinct sizes, shapes, and functions. For example, most flowering plants contain specialized petal conical epidermal cells that are thought to attract pollinators and influence light capture and reflectance, but the molecular mechanisms controlling conical cell shaping remain unclear. Here, through a genetic screen in Arabidopsis thaliana, we demonstrated that loss-of-function mutations in ANGUSTIFOLIA (AN), which encodes for a homolog of mammalian CtBP/BARs, displayed conical cells phenotype with wider tip angles, correlating with increased accumulation of reactive oxygen species (ROS). We further showed that exogenously supplied ROS generated similar conical cell phenotypes as the an mutants. Moreover, reduced endogenous ROS levels resulted in deceased tip sharpening of conical cells. Furthermore, through enhancer screening, we demonstrated that mutations in katanin (KTN1) enhanced conical cell phenotypes of the an-t1 mutants. Genetic analyses showed that AN acted in parallel with KTN1 to control conical cell shaping. Both increased or decreased ROS levels and mutations in AN suppressed microtubule organization into well-ordered circumferential arrays. We demonstrated that the AN-ROS pathway jointly functioned with KTN1 to modulate microtubule ordering, correlating with the tip sharpening of conical cells. Collectively, our findings revealed a mechanistic insight into ROS homeostasis regulation of microtubule organization and conical cell shaping.

Spatio-temporal orientation of microtubules controls conical cell shape in Arabidopsis thaliana petals.

The physiological functions of epidermal cells are largely determined by their diverse morphologies. Most flowering plants have special conical-shaped petal epidermal cells that are thought to influence light capture and reflectance, and provide pollinator grips, but the molecular mechanisms controlling conical cell shape remain largely unknown. Here, we developed a live-confocal imaging approach to quantify geometric parameters of conical cells in Arabidopsis thaliana (A. thaliana). Through genetic screens, we identified katanin (KTN1) mutants showing a phenotype of decreased tip sharpening of conical cells. Furthermore, we demonstrated that SPIKE1 and Rho of Plants (ROP) GTPases were required for the final shape formation of conical cells, as KTN1 does. Live-cell imaging showed that wild-type cells exhibited random orientation of cortical microtubule arrays at early developmental stages but displayed a well-ordered circumferential orientation of microtubule arrays at later stages. By contrast, loss of KTN1 prevented random microtubule networks from shifting into well-ordered arrays. We further showed that the filamentous actin cap, which is a typical feature of several plant epidermal cell types including root hairs and leaf trichomes, was not observed in the growth apexes of conical cells during cell development. Moreover, our genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our results provide a novel imaging approach for studying petal conical cell morphogenesis and suggest that the spatio-temporal organization of microtubule arrays plays crucial roles in controlling conical cell shape.

Contribution of heavy metals to toxicity of coal combustion related fine particulate matter (PM2.5) in Caenorhabditis elegans with wild-type or susceptible genetic background.

Contribution of chemical components in coal combustion related fine particulate matter (PM2.5) to its toxicity is largely unclear. We focused on heavy metals in PM2.5 to investigate their contribution to toxicity formation in Caenorhabditis elegans. Among 8 heavy metals examined (Fe, Zn, Pb, As, Cd, Cr, Cu, and Ni), Pb, Cr, and Cu potentially contributed to PM2.5 toxicity in wild-type nematodes. Combinational exposure to any two of these three heavy metals caused higher toxicity than exposure to Pb, Cr, or Cu alone. Toxicity from the combinational exposure to Pb, Cr, and Cu at the examined concentrations was higher than exposure to PM2.5 (100 mg/L). Moreover, mutation of sod-2 or sod-3 gene encoding Mn-SOD increased susceptibility in nematodes exposed to Fe, Zn, or Ni, although Fe, Zn, or Ni at the examined concentration did not lead to toxicity in wild-type nematodes. Our results highlight the potential contribution of heavy metals to PM2.5 toxicity in environmental organisms.