Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.

The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

The cell cortex-localized protein CHDP-1 is required for dendritic development and transport in C. elegans neurons.

Cortical actin, a thin layer of actin network underneath the plasma membranes, plays critical roles in numerous processes, such as cell morphogenesis and migration. Neurons often grow highly branched dendrite morphologies, which is crucial for neural circuit assembly. It is still poorly understood how cortical actin assembly is controlled in dendrites and whether it is critical for dendrite development, maintenance and function. In the present study, we find that knock-out of C. elegans chdp-1, which encodes a cell cortex-localized protein, causes dendrite formation defects in the larval stages and spontaneous dendrite degeneration in adults. Actin assembly in the dendritic growth cones is significantly reduced in the chdp-1 mutants. PVD neurons sense muscle contraction and act as proprioceptors. Loss of chdp-1 abolishes proprioception, which can be rescued by expressing CHDP-1 in the PVD neurons. In the high-ordered branches, loss of chdp-1 also severely affects the microtubule cytoskeleton assembly, intracellular organelle transport and neuropeptide secretion. Interestingly, knock-out of sax-1, which encodes an evolutionary conserved serine/threonine protein kinase, suppresses the defects mentioned above in chdp-1 mutants. Thus, our findings suggest that CHDP-1 and SAX-1 function in an opposing manner in the multi-dendritic neurons to modulate cortical actin assembly, which is critical for dendrite development, maintenance and function.

Ni Single-Atom Bual Catalytic Electrodes for Long Life and High Energy Efficiency Zinc-Iodine Batteries.

Zinc-iodine batteries (Zn-I2) are extremely attractive as the safe and cost-effective scalable energy storage system in the stationary applications. However, the inefficient redox kinetics and "shuttling effect" of iodine species result in unsatisfactory energy efficiency and short cycle life, hindering their commercialization. In this work, Ni single atoms highly dispersed on carbon fibers is designed and synthesized as iodine anchoring sites and dual catalysts for Zn-I2 batteries, and successfully inhibit the iodine species shuttling and boost dual reaction kinetics. Theoretical calculations indicate that the reinforced d-p orbital hybridization and charge interaction between Ni single-atoms and iodine species effectively enhance the confinement of iodine species. Ni single-atoms also accelerate the iodine conversion reactions with tailored bonding structure of I─I bonds and reduced energy barrier for the dual conversion of iodine species. Consequently, the high-rate performance (180 mAh g-1 at 3 A g-1), cycling stability (capacity retention of 74% after 5900 cycles) and high energy efficiency (90% at 3 A g-1) are achieved. The work provides an effective strategy for the development of iodine hosts with high catalytic activity for Zn-I2 batteries.

Two-Color CRISPR Imaging Reveals Dynamics of Herpes Simplex Virus 1 Replication Compartments and Virus-Host Interactions.

Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. CRISPR-assisted single viral genome tracking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato10x and dSpCas9-GFP14x were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. IMPORTANCE Herpes simplex virus 1 (HSV-1), a representative of the family Herpesviridae, is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.

Mechanical Force Induced Self-Assembly of Chinese Herbal Hydrogel with Synergistic Effects of Antibacterial Activity and Immune Regulation for Wound Healing.

Skin wounds, especially infected chronic wounds, have attracted worldwide attention due to the high prevalence and poor treatment outcomes. Hydrogel dressings with antibacterial ability and immune regulation property are urgently required. Herein, inspired by the grinding treatment of traditional Chinese medicine, mechanical force is introduced to promote the effective molecular collision and accelerate the self-assembly of chitosan (CS) and puerarin (PUE) for fabricating Chinese-herb-based hydrogels. The antibacterial rate of CS@PUE (C@P) hydrogel is more than 95%, and the wound closed rate is twice that of the control group. Interestingly, the rational design of C@P hydrogels with different PUE ratios enables a refined control over hydrogel formation, nanofiber appearance, viscoelastic, physicochemical, and biological properties. The extraordinary antibacterial ability of C@P hydrogels may originate from the nanofiber structure and the improved zeta potential on account of the orientation of amino groups in CS . Thus, the synergistically antibacterial and immune regulation properties of C@P hydrogels kill bacteria and relieve inflammation in the wound bed, ensuring the anti-infection effect, and boosting wound healing. In addition to providing a universal mechanosynthesis of PUE-based hydrogel for wound healing, this finding is expected to increase the attention paid to Chinese herbal medicines in the construction of biomaterials.

Low-voltage and fast-response polymer-stabilized blue-phase liquid crystals achieved using a new organosilicone monomer.

In this paper, two types of polymer-stabilized blue-phase liquid crystals (PS-BPLCs) with different monomers were designed and prepared. The morphology, temperature range and electro-optical properties of the blue phases were studied and discussed. The temperature range of both types of PS-BPLC is greater than 110 °C, and both samples can be stabilized well at room temperature. The organosilicone monomer 3-methacryloxypropyltrimethoxysilane (KH570), which contains double bonds, was introduced to a blue-phase system for the first time. Regarding the electro-optical performance, the on-state voltage of the PS-BPLCs with the KH570 monomer is reduced to 30 V compared with traditional C12A monomer systems in which the on-state voltage is 75 V at 458 nm. Meanwhile, a fast response and suppressed hysteresis are obtained. These results are helpful to the application of displays and photonic devices.

TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

Correlation analysis framework for localization-based superresolution microscopy.

Superresolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based superresolution images using distance histograms. We demonstrate the application of this concept in multiple scenarios, including image alignment, tracking of diffusing molecules, as well as for quantification of colocalization, showing its superior performance over existing approaches.

A20 Restores Impaired Intestinal Permeability and Inhibits Th2 Response in Mice with Colitis.

BACKGROUND/AIMS: The etiology of inflammatory bowel disease is multifactorial and still obscure. The protective role of ubiquitin E3 ligase A20 (A20) in colitis needs to be further elucidated. This study aimed to investigate whether A20 exogenous administration restored impaired intestinal permeability and inhibited T helper (Th)2 response in mice with colitis. METHODS: The effect of A20 overexpression in colonic mucosa on epithelial barrier function and T cell differentiation was evaluated in mice with dextran sulfate sodium (DSS)-induced chronic colitis. RESULTS: A20 rectal treatment alleviated DSS-induced chronic colitis and restored impaired intestinal permeability. Oral challenge with 2% DSS elicited a Th2-type response in mice with colitis, and A20 rectal treatment inhibited CD4+ interleukin (IL)-4+ T cell differentiation and proliferation. In addition, the RNA expressions of Th2-related costimulatory molecular T-cell immunoglobulin and mucin domain (TIM)-1 and IL-4 were suppressed, while thrombospondin (TSP)-1 and interferon (IFN)-γ expressions were upregulated, after A20 rectal administration. CONCLUSION: A20 rectal treatment restores impaired intestinal permeability and inhibits activated Th2 cell response in mice with colitis.

ATL3 gene mutation in a Chinese family with hereditary sensory neuropathy type 1F.

Hereditary sensory neuropathy (HSN) comprises a group of progressive peripheral neuropathies predominantly affecting the sensory nerves. To date, two different ATL3 gene mutations have been reported to be responsible for HSN type 1F (HSN1F). Here, we report a family in which the members presented numbness of the lower limbs and recurrent foot ulceration. Symptoms of foot ulcers disappeared in the years after onset, which suggests that the family members showed benign and mild symptoms compared with the affected patients reported previously. Laboratory examinations and electrophysiological data suggested axonal degeneration of the peripheral sensory nerves, while motor neurons were not involved. Exome sequencing revealed the previously reported c.C1013G (p.Pro338Arg) mutation of the ATL3 gene. This is the first report of ATL3 mutation in Chinese patients with HSN. Cells expressing mutant ATL3 exhibited disruption of the endoplasmic reticulum network, suggesting a dominant-negative effect. There was no significant difference in the expression of the endoplasmic reticulum stress marker binding immunoglobulin protein (BiP) between cells expressing wild-type or mutant ATL3. Further studies are required to ascertain the relevance of the changes in endoplasmic reticulum morphology to axonal degeneration of sensory nerves.

Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon.

Acidovorax citrulli is the causal agent of bacterial fruit blotch, a serious threat to commercial watermelon and melon crop production worldwide. Ferric uptake regulator (Fur) is a global transcription factor that affects a number of virulence-related functions in phytopathogenic bacteria; however, the role of furA has not been determined for A. citrulli. Hence, we constructed an furA deletion mutant and a corresponding complement in the background of A. citrulli strain xlj12 to investigate the role of the gene in siderophore production, concentration of intracellular Fe2+, bacterial sensitivity to hydrogen peroxide, biofilm formation, swimming motility, hypersensitive response induction, and virulence on melon seedlings. The A. citrulli furA deletion mutant displayed increased siderophore production, intracellular Fe2+ concentration, and increased sensitivity to hydrogen peroxide. In contrast, biofilm formation, swimming motility, and virulence on melon seedlings were significantly reduced in the furA mutant. As expected, complementation of the furA deletion mutant restored all phenotypes to wild-type levels. In accordance with the phenotypic results, the expression levels of bfrA and bfrB that encode bacterioferritin, sodB that encodes iron/manganese superoxide dismutase, fliS that encodes a flagellar protein, hrcN that encodes the type III secretion system (T3SS) ATPase, and hrcC that encodes the T3SS outer membrane ring protein were significantly downregulated in the A. citrulli furA deletion mutant. In addition, the expression of feo-related genes and feoA and feoB was significantly upregulated in the furA mutant. Overall, these results indicated that, in A. citrulli, FurA contributes to the regulation of the iron balance system, and affects a variety of virulence-related traits.

Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci.

In order to elucidate the functional organization of the genome, it is vital to directly visualize the interactions between genomic elements in living cells. For this purpose, we engineered the Cas9 protein from Staphylococcus aureus (SaCas9) for the imaging of endogenous genomic loci, which showed a similar robustness and efficiency as previously reported for Streptococcus pyogenes Cas9 (SpCas9). Imaging readouts allowed us to characterize the DNA-binding activity of SaCas9 and to optimize its sgRNA scaffold. Combining SaCas9 and SpCas9, we demonstrated two-color CRISPR imaging with the capability to resolve genomic loci spaced by <300 kb. Combinatorial color-mixing further enabled us to code multiple genomic elements in the same cell. Our results highlight the potential of combining SpCas9 and SaCas9 for multiplexed CRISPR-Cas9 applications, such as imaging and genome engineering.

Formulating compatible non-flammable electrolyte for lithium-ion batteries with ethoxy (pentafluoro) cyclotriphosphazene.

Lithium (Li) ion batteries have played a great role in modern society as being extensively used in commercial electronic products, electric vehicles, and energy storage systems. However, battery safety issues have gained growing concerns as there might be thermal runaway, fire or even explosion under external abuse. To tackle these safety issues, developing non-flammable electrolytes is a promising strategy. However, the balance between the flame-retarding effect and the electrochemical performance of electrolytes remains a great challenge. Herein, we evaluate the function of ethoxy (pentafluoro) cyclotriphosphazene (PFPN) as an effective flame-retarding additive for lithium-ion batteries. The flammability of electrolytes is greatly suppressed with the introduction of a small amount of PFPN. Moreover, PFPN exhibited excellent compatibility with LiFePO4 (LFP) cathode and graphite (Gr) anode, the electrochemical performances of LFP|Li and Gr|Li half cells are virtually unaffected. Scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS) reveal the stable interphase between PFPN-containing electrolyte and LFP and Gr electrodes. Fourier transform infrared spectroscopy (FT-IR), Raman and nuclear magnetic resonance (NMR) spectra demonstrate the introduction of PFPN only exhibits negligible influence on the solvation structure of electrolyte. Benefiting from these merits of PFPN, the LFP|Gr cell shows desirable long-term cycling performance, which demonstrates great potential for practical application.

Endocytic sorting and recycling require membrane phosphatidylserine asymmetry maintained by TAT-1/CHAT-1.

Endocytic sorting is achieved through the formation of morphologically and functionally distinct sub-domains within early endosomes. Cargoes destined for recycling are sorted to and transported through newly-formed tubular membranes, but the processes that regulate membrane tubulation are poorly understood. Here, we identified a novel Caenorhabditis elegans Cdc50 family protein, CHAT-1, which acts as the chaperone of the TAT-1 P4-ATPase to regulate membrane phosphatidylserine (PS) asymmetry and endocytic transport. In chat-1 and tat-1 mutants, the endocytic sorting process is disrupted, leading to defects in both cargo recycling and degradation. TAT-1 and CHAT-1 colocalize to the tubular domain of the early endosome, the tubular endocytic recycling compartment (ERC), and the recycling endosome where PS is enriched on the cytosolic surface. Loss of tat-1 and chat-1 function disrupts membrane PS asymmetry and abrogates the tubular membrane structure. Our data suggest that CHAT-1 and TAT-1 maintain membrane phosphatidylserine asymmetry, thus promoting membrane tubulation and regulating endocytic sorting and recycling.

Ribo-On and Ribo-Off tools using a self-cleaving ribozyme allow manipulation of endogenous gene expression in C. elegans.

Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between "on" and "off" states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3' untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP-flanked transcriptional termination signal and ribozyme is inserted into the 3' UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP-flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes.

Chitosan@Puerarin hydrogel for accelerated wound healing in diabetic subjects by miR-29ab1 mediated inflammatory axis suppression.

Wound healing is one of the major global health concerns in patients with diabetes. Overactivation of pro-inflammatory M1 macrophages is associated with delayed wound healing in diabetes. miR-29ab1 plays a critical role in diabetes-related macrophage inflammation. Hence, inhibition of inflammation and regulation of miR-29 expression have been implicated as new points for skin wound healing. In this study, the traditional Chinese medicine, puerarin, was introduced to construct an injectable and self-healing chitosan@puerarin (C@P) hydrogel. The C@P hydrogel promoted diabetic wound healing and accelerated angiogenesis, which were related to the inhibition of the miR-29 mediated inflammation response. Compared to healthy subjects, miR-29a and miR-29b1 were ectopically increased in the skin wound of the diabetic model, accompanied by upregulated M1-polarization, and elevated levels of IL-1ß and TNF-α. Further evaluations by miR-29ab1 knockout mice exhibited superior wound healing and attenuated inflammation. The present results suggested that miR-29ab1 is essential for diabetic wound healing by regulating the inflammatory response. Suppression of miR-29ab1 by the C@P hydrogel has the potential for improving medical approaches for wound repair.

Illuminating the Live-Cell Dynamics of Hepatitis B Virus Covalently Closed Circular DNA Using the CRISPR-Tag System.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major obstacle to curing chronic hepatitis B (CHB). Current cccDNA detection methods are mostly based on biochemical extraction and bulk measurements. They nevertheless generated a general sketch of its biological features. However, an understanding of the spatiotemporal features of cccDNA is still lacking. To achieve this, we established a system combining CRISPR-Tag and recombinant HBV minicircle technology to visualize cccDNA at single-cell level in real time. Using this system, we found that the observed recombinant cccDNA (rcccDNA) correlated quantitatively with its active transcripts when a low to medium number of foci (<20) are present, but this correlation was lost in cells harboring high copy numbers (≥20) of rcccDNA. The disruption of HBx expression seems to displace cccDNA from the dCas9-accessible region, while HBx complementation restored the number of observable cccDNA foci. This indicated regulation of cccDNA accessibility by HBx. Second, observable HBV and duck HBV (DHBV) cccDNA molecules are substantially lost during cell division, and the remaining ones were distributed randomly to daughter cells. In contrast, Kaposi's sarcoma-associated herpesvirus (KSHV)-derived episomes can be retained in a LANA (latency-associated nuclear antigen)-dependent manner. Last, the dynamics of rcccDNA episomes in nuclei displayed confined diffusion at short time scales, with directional transport over longer time scales. In conclusion, this system enables the study of physiological kinetics of cccDNA at the single-cell level. The differential accessibility of rcccDNA to dCas9 under various physiological conditions may be exploited to elucidate the complex transcriptional and epigenetic regulation of the HBV minichromosome. IMPORTANCE Understanding the formation and maintenance of HBV cccDNA has always been a central issue in the study of HBV pathobiology. However, little progress has been made due to the lack of robust assay systems and its resistance to genetic modification. Here, a live-cell imaging system by grafting CRISPR-Tag into the recombinant cccDNA was established to visualize its molecular behavior in real time. We found that the accessibility of rcccDNA to dCas9-based imaging is related to HBx-regulated mechanisms. We also confirmed the substantial loss of observable rcccDNA in one-round cell division and random distribution of the remaining molecules. Molecular dynamics analysis revealed the confined movement of the rcccDNA episome, suggesting its juxtaposition to chromatin domains. Overall, this novel system offers a unique platform to investigate the intranuclear dynamics of cccDNA within live cells.

Real-time imaging of RNA polymerase I activity in living human cells.

RNA polymerase I (Pol I) synthesizes about 60% of cellular RNA by transcribing multiple copies of the ribosomal RNA gene (rDNA). The transcriptional activity of Pol I controls the level of ribosome biogenesis and cell growth. However, there is currently a lack of methods for monitoring Pol I activity in real time. Here, we develop LiveArt (live imaging-based analysis of rDNA transcription) to visualize and quantify the spatiotemporal dynamics of endogenous ribosomal RNA (rRNA) synthesis. LiveArt reveals mitotic silencing and reactivation of rDNA transcription, as well as the transcriptional kinetics of interphase rDNA. Using LiveArt, we identify SRFBP1 as a potential regulator of rRNA synthesis. We show that rDNA transcription occurs in bursts and can be altered by modulating burst duration and amplitude. Importantly, LiveArt is highly effective in the screening application for anticancer drugs targeting Pol I transcription. These approaches pave the way for a deeper understanding of the mechanisms underlying nucleolar functions.

Puerarin@Chitosan composite for infected bone repair through mimicking the bio-functions of antimicrobial peptides.

It is important to eliminate lipopolysaccharide (LPS) along with killing bacteria in periprosthetic joint infection (PJI) therapy for promoting bone repair due to its effect to regulate macrophages response. Although natural antimicrobial peptides (AMPs) offer a good solution, the unknown toxicity, high cost and exogenetic immune response hamper their applications in clinic. In this work, we fabricated a nanowire-like composite material, named P@C, by combining chitosan and puerarin via solid-phase reaction, which can finely mimic the bio-functions of AMPs. Chitosan, serving as the bacteria membrane puncture agent, and puerarin, serving as the LPS target agent, synergistically destroy the bacterial membrane structure and inhibit its recovery, thus endowing P@C with good antibacterial property. In addition, P@C possesses good osteoimmunomodulation due to its ability of LPS elimination and macrophage differentiation modulation. The in vivo results show that P@C can inhibit the LPS induced bone destruction in the Escherichia coli infected rat. P@C exhibits superior bone regeneration in Escherichia coli infected rat due to the comprehensive functions of its superior antibacterial property, and its ability of LPS elimination and immunomodulation. P@C can well mimic the functions of AMPs, which provides a novel and effective method for treating the PJI in clinic.