Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome, bioRxiv, 2019-03-06
AbstractCRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse microbiomes.
biorxiv microbiology 0-100-users 2019The Trichoplax microbiome the simplest animal lives in an intimate symbiosis with two intracellular bacteria, bioRxiv, 2019-03-06
Summary paragraphPlacozoa is an enigmatic phylum of simple, microscopic, marine metazoans. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax H2 lives in symbiosis with two intracellular bacteria. One symbiont forms a new genus in the Midichloriaceae (Rickettsiales) and has a genomic repertoire similar to that of rickettsial parasites, but does not appear to express key genes for energy parasitism. Correlative microscopy and 3-D electron tomography revealed that this symbiont resides in an unusual location, the rough endoplasmic reticulum of its host’s internal fiber cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations. This symbiont lives in the ventral epithelial cells of Trichoplax, likely metabolizes algal lipids digested by its host, and has the capacity to supplement the placozoan’s nutrition. Our study shows that even the simplest animals known have evolved highly specific and intimate associations with symbiotic, intracellular bacteria, and highlights that symbioses with microorganisms are a basal trait of animal life.
biorxiv microbiology 100-200-users 2019TheTrichoplaxmicrobiome the simplest animal lives in an intimate symbiosis with two intracellular bacteria, bioRxiv, 2019-03-06
Summary paragraphPlacozoa is an enigmatic phylum of simple, microscopic, marine metazoans. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoanTrichoplaxH2 lives in symbiosis with two intracellular bacteria. One symbiont forms a new genus in the Midichloriaceae (Rickettsiales) and has a genomic repertoire similar to that of rickettsial parasites, but does not appear to express key genes for energy parasitism. Correlative microscopy and 3-D electron tomography revealed that this symbiont resides in an unusual location, the rough endoplasmic reticulum of its host’s internal fiber cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations. This symbiont lives in the ventral epithelial cells ofTrichoplax, likely metabolizes algal lipids digested by its host, and has the capacity to supplement the placozoan’s nutrition. Our study shows that even the simplest animals known have evolved highly specific and intimate associations with symbiotic, intracellular bacteria, and highlights that symbioses with microorganisms are a basal trait of animal life.
biorxiv microbiology 100-200-users 2019A robust and efficient method for Mendelian randomization with hundreds of genetic variants unravelling mechanisms linking HDL-cholesterol and coronary heart disease, bioRxiv, 2019-03-05
Mendelian randomization (MR) investigations with large numbers of genetic variants are becoming increasingly common. However, the reliability of findings from a MR investigation is dependent on the validity of the genetic variants as instrumental variables. We developed a method to identify groups of genetic variants with similar causal effect estimates, which may represent distinct mechanisms by which the risk factor influences the outcome. Our contamination mixture method is a robust and efficient method for valid MR in the presence of invalid IVs. Compared to other robust methods, our method had the lowest mean squared error across a range of realistic scenarios. The method is fast and efficient, and can perform analysis with hundreds of variants in a fraction of a second. In a MR analysis for high-density lipoprotein (HDL) cholesterol and coronary heart disease (CHD) risk, the method identified 11 variants associated with increased HDL-cholesterol, decreased triglyceride levels, and decreased CHD risk that had the same directions of associations with platelet distribution width and other blood cell traits, suggesting a shared mechanism linking lipids and CHD risk relating to platelet aggregation.
biorxiv genetics 0-100-users 2019About samples, giving examples Optimized Single Molecule Localization Microscopy, bioRxiv, 2019-03-05
AbstractSuper-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.
biorxiv cell-biology 200-500-users 2019Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin, bioRxiv, 2019-03-05
ABSTRACTThree-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function, however many of its physical features at the single-cell level remain underexplored. Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ∼200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in post-replicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.
biorxiv genomics 100-200-users 2019