MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

Cardiac expression of a gain-of-function α5-integrin results in perinatal lethality

2001 ◽  
Vol 280 (1) ◽  
pp. H361-H367 ◽  
Author(s):  
Maria L. Valencik ◽  
John A. McDonald
Keyword(s):  
Cardiac Development ◽  
Wild Type ◽  
Integrin Receptors ◽  
Gain Of Function ◽  
Transgenic Expression ◽  
Physiological Regulation ◽  
Myocyte Differentiation ◽  
Perinatal Lethality ◽  
And Function

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. α5β1-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of α5β1-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human α5-integrin in cardiomyocytes: the full-length wild-type α5-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated α5-1-integrin. Overexpression of the wild-type α5-integrin has no detectable adverse effects in the mouse, whereas expression of α5-1-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.

scholarly journals Phyletic distribution and diversification of the Phage Shock Protein stress response system in bacteria and archaea

2021 ◽  
Author(s):  
Philipp F. Popp ◽  
Vadim M. Gumerov ◽  
Ekaterina P. Andrianova ◽  
Lisa Bewersdorf ◽  
Thorsten Mascher ◽  
...  
Keyword(s):  
Stress Response ◽  
Large Scale ◽  
Cell Envelope ◽  
Model Organisms ◽  
Microbial World ◽  
Natural Classification ◽  
Response System ◽  
Core Proteins ◽  
Envelope Stress

AbstractThe bacterial cell envelope is an essential structure that protects the cell from environmental threats, while simultaneously serving as communication interface and diffusion barrier. Therefore, maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protection networks that connect stress perception, transmembrane signal transduction and mediation of cellular responses upon cell envelope stress. The phage shock protein (PSP) stress response is one of such conserved protection networks. Most of the knowledge about the Psp response comes from studies in the Gram-negative model bacterium, Escherichia coli where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of Proteobacteria, Actinobacteria, and Firmicutes; however, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and demonstrated that the PSP system deviates dramatically from the proteobacterial prototype. Two of its core proteins, PspA and PspC, have been integrated in various (often phylum-specifically) conserved protein networks during evolution. Based on protein sequence and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system of PSP networks in bacteria and archaea. We performed a comprehensive in vivo protein interaction screen for the PSP network newly identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected PSP response system, illustrating the validity of our approach. Our study highlights the diversity of PSP organization and function across many bacterial and archaeal phyla and will serve as foundation for future studies of this envelope stress response beyond model organisms.

scholarly journals iProteinDB: an integrative database of Drosophila post-translational modifications

2018 ◽  
Author(s):  
Yanhui Hu ◽  
Richelle Sopko ◽  
Verena Chung ◽  
Romain A. Studer ◽  
Sean D. Landry ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Large Scale ◽  
Model Organisms ◽  
General Strategy ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Sites ◽  
Evolutionarily Conserved ◽  
And Function

AbstractPost-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

scholarly journals Large-Scale Chronically Implantable Precision Motorized Microdrive Array for Freely Behaving Animals

2008 ◽  
Vol 100 (4) ◽  
pp. 2430-2440 ◽  
Author(s):  
Jun Yamamoto ◽  
Matthew A. Wilson
Keyword(s):  
Single Unit ◽  
Large Scale ◽  
Neural Circuits ◽  
Brain Regions ◽  
Unit Recording ◽  
Chronic Recording ◽  
Large Numbers ◽  
Freely Behaving

Multiple single-unit recording has become one of the most powerful in vivo electro-physiological techniques for studying neural circuits. The demand has been increasing for small and lightweight chronic recording devices that allow fine adjustments to be made over large numbers of electrodes across multiple brain regions. To achieve this, we developed precision motorized microdrive arrays that use a novel motor multiplexing headstage to dramatically reduce wiring while preserving precision of the microdrive control. Versions of the microdrive array were chronically implanted on both rats (21 microdrives) and mice (7 microdrives), and relatively long-term recordings were taken.

scholarly journals Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions

2008 ◽  
Vol 295 (5) ◽  
pp. L744-L755 ◽  
Author(s):  
James West ◽  
Julie Harral ◽  
Kirk Lane ◽  
Yupu Deng ◽  
Brian Ickes ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Gene Array ◽  
Vascular Lesions ◽  
Tail Domain ◽  
Small Vessels ◽  
Morphogenetic Protein ◽  
Large Numbers ◽  
Proliferation And Apoptosis ◽  
And Function

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO7-BMPR2R899X mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO7-BMPR2R899X mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.

scholarly journals Targeted capture of complete coding regions across divergent species

2017 ◽  
Author(s):  
Ryan K Schott ◽  
Bhawandeep Panesar ◽  
Daren C Card ◽  
Matthew Preston ◽  
Todd A Castoe ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Large Scale ◽  
Functional Studies ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Phylogenetic Studies ◽  
Large Numbers ◽  
Capture Method ◽  
Targeted Capture ◽  
And Function

AbstractDespite continued advances in sequencing technologies, there is a need for methods that can efficiently sequence large numbers of genes from diverse species. One approach to accomplish this is targeted capture (hybrid enrichment). While these methods are well established for genome resequencing projects, cross-species capture strategies are still being developed and generally focus on the capture of conserved regions, rather than complete coding regions from specific genes of interest. The resulting data is thus useful for phylogenetic studies, but the wealth of comparative data that could be used for evolutionary and functional studies is lost. Here we design and implement a targeted capture method that enables recovery of complete coding regions across broad taxonomic scales. Capture probes were designed from multiple reference species and extensively tiled in order to facilitate cross-species capture. Using novel bioinformatics pipelines we were able to recover nearly all of the targeted genes with high completeness from species that were up to 200 myr divergent. Increased probe diversity and tiling for a subset of genes had a large positive effect on both recovery and completeness. The resulting data produced an accurate species tree, but importantly this same data can also be applied to studies of molecular evolution and function that will allow researchers to ask larger questions in broader phylogenetic contexts. Our method demonstrates the utility of cross-species approaches for the capture of full length coding sequences, and will substantially improve the ability for researchers to conduct large-scale comparative studies of molecular evolution and function.

scholarly journals Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

2020 ◽  
Author(s):  
R Christian McDonald ◽  
Matthew J Schott ◽  
Temitope A Idowu ◽  
Peter J Lyons
Keyword(s):  
Enzyme Activity ◽  
Large Scale ◽  
Structure And Function ◽  
Activation Mechanism ◽  
Marker Genes ◽  
Synthetic Lethal ◽  
Fungal Process ◽  
And Function

Abstract Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

scholarly journals Cloning and Characterization of a Novel Subunit of Protein Serine/Threonine Phosphatase 4 from Mesangial Cells

2001 ◽  
Vol 12 (12) ◽  
pp. 2601-2608 ◽  
Author(s):  
Takehiko Wada ◽  
Toshio Miyata ◽  
Reiko Inagi ◽  
Masaomi Nangaku ◽  
Masako Wagatsuma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Cultured Cells ◽  
Mesangial Cells ◽  
Spindle Pole ◽  
Regulatory Subunit ◽  
Glomerular Diseases ◽  
Regulatory Subunits ◽  
Novel Approach

ABSTRACT. Mesangial cells play an important role in maintaining glomeruli structure and function and in the pathogenesis of glomerular diseases. With a novel approach using a rapid large-scale DNA sequencing strategy and computerized data processing, a new human gene, PP4Rmegwas cloned. The full-length cDNA clone of human PP4Rmegcoded for a novel 950-amino acid protein, which was similar to a subunit of protein serine/threonine phosphatase 4 (PP4). Recombinant PP4Rmegproduced in COS-7 cells bound to the catalytic subunit of PP4. PP4Rmegis therefore structurally and functionally related to the recently reported regulatory subunit of PP4, PP4R1. Amino acid sequence analysis of rat PP4Rmeghomologue revealed that the sequences were well conserved between human and rat (86.3% identity). Northern blot analyses of human tissues and cultured cells demonstrated that the regulatory subunits were expressed abundantly in human cultured mesangial cells, although their expression was relatively ubiquitous.In situhybridization studies in normal human renal tissues confirmed their expression in glomeruliin vivo. The expression was upregulated in glomeruli of anti-Thy1 glomerulonephritis rats before mesangial proliferation. These data demonstrate that PP4Rmegis a novel regulatory subunit of PP4, which is expressed ubiquitously but abundantly in mesangial cells. Its pathophysiologic role in mesangial cells and glomerulus remains unknown. As PP4 is an essential protein for nucleation, growth, and stabilization of microtubules at centrosomes/spindle pole bodies during cell division, PP4Rmegmay play a role in regulation of mitosis in mesangial cells.

scholarly journals From single nuclei to whole genome assemblies

2019 ◽  
Author(s):  
Merce Montoliu-Nerin ◽  
Marisol Sánchez-García ◽  
Claudia Bergin ◽  
Manfred Grabherr ◽  
Barbara Ellis ◽  
...  
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Genomic Data ◽  
Life Cycles ◽  
Genomic Research ◽  
Metagenomic Data ◽  
Model Organisms ◽  
Genomic Study ◽  
And Function ◽  
Genome Assemblies

SummaryA large proportion of Earth's biodiversity constitutes organisms that cannot be cultured, have cryptic life-cycles and/or live submerged within their substrates1–4. Genomic data are key to unravel both their identity and function5. The development of metagenomic methods6,7 and the advent of single cell sequencing8–10 have revolutionized the study of life and function of cryptic organisms by upending the need for large and pure biological material, and allowing generation of genomic data from complex or limited environmental samples. Genome assemblies from metagenomic data have so far been restricted to organisms with small genomes, such as bacteria11, archaea12 and certain eukaryotes13. On the other hand, single cell technologies have allowed the targeting of unicellular organisms, attaining a better resolution than metagenomics8,9,14–16, moreover, it has allowed the genomic study of cells from complex organisms one cell at a time17,18. However, single cell genomics are not easily applied to multicellular organisms formed by consortia of diverse taxa, and the generation of specific workflows for sequencing and data analysis is needed to expand genomic research to the entire tree of life, including sponges19, lichens3,20, intracellular parasites21,22, and plant endophytes23,24. Among the most important plant endophytes are the obligate mutualistic symbionts, arbuscular mycorrhizal (AM) fungi, that pose an additional challenge with their multinucleate coenocytic mycelia25. Here, the development of a novel single nuclei sequencing and assembly workflow is reported. This workflow allows, for the first time, the generation of reference genome assemblies from large scale, unbiased sorted, and sequenced AM fungal nuclei circumventing tedious, and often impossible, culturing efforts. This method opens infinite possibilities for studies of evolution and adaptation in these important plant symbionts and demonstrates that reference genomes can be generated from complex non-model organisms by isolating only a handful of their nuclei.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

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