Project description:Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis.
Project description:Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.
Project description:Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.
Project description:A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.
Project description:Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.
Project description:Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.
Project description:Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.
Project description:Single genomic loci are often related to specific cellular functions, genetic diseases, or pathogenic infections. Visualization of single genomic loci in live human cells is currently of great interest, yet it remains challenging. Here, we describe a strategy for live cell imaging of single genomic loci by combining transcription activator-like effectors (TALEs) with a quantum dot labelling technique. We design and select a pair of TALEs that specifically target HIV-1 proviral DNA sequences, and use bioorthogonal ligation reactions to label them with different colour quantum dots (QDs). These QD-labelled TALEs are able to enter the cell nucleus to provide fluorescent signals to identify single gene loci. Based on the co-localization of the pair of different coloured QD-labelled TALEs, we determine and map single-copy HIV-1 provirus loci in human chromosomes in live host cells.
Project description:The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based on the CRISPR-dPspCas13b system with fluorescent RNA aptamers in sgRNA (CasFAS) in live cells. Using modified sgRNA attached to fluorescent RNA aptamers that showed reduced background fluorescence, this approach provides a simple, sensitive way to image and track endogenous RNA with high accuracy and efficiency. In addition, color switching can be easily achieved by changing the fluorogenic dye analogues in living cells through user-friendly washing and restaining operations. CasFAS is compatible with orthogonal fluorescent aptamers, such as Broccoli and Pepper, enabling multiple colors RNA labeling or intracellular RNA-RNA interaction imaging. Finally, the visualization of severe fever with thrombocytopenia syndrome virus (SFTSV) was achieved by CasFAS, which may facilitate further studies on this virus.
Project description:Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.