Miniaturizing 3D assay for high-throughput drug and genetic screens for small patient-derived tumor samples (Conference Presentation)

2017 ◽  
Author(s):  
Asaf Rotem ◽  
Levi Garraway ◽  
Mei-Ju Su ◽  
Anindita Basu ◽  
Aviv Regev ◽  
...  
Keyword(s):  
High Throughput ◽  
Genetic Screens ◽  
Small Patient

scholarly journals High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding

2019 ◽  
Vol 218 (8) ◽  
pp. 2797-2811 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Fluorescent Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Cell Type ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

scholarly journals Matrix linear models for high-throughput chemical genetic screens

2018 ◽  
Author(s):  
Jane W. Liang ◽  
Robert J. Nichols ◽  
Śaunak Sen
Keyword(s):  
High Throughput ◽  
Linear Models ◽  
Natural Extension ◽  
Attractive Alternative ◽  
Genetic Screens ◽  
Computationally Efficient ◽  
Model Framework ◽  
Chemical Genetic ◽  
Link Type ◽  
Univariate Approach

AbstractWe develop a flexible and computationally efficient approach for analysing high throughput chemical genetic screens. In such screens, a library of genetic mutants is phenotyped in a large number of stresses. The goal is to detect interactions between genes and stresses. Typically, this is achieved by grouping the mutants and stresses into categories, and performing modified t-tests for each combination. This approach does not have a natural extension if mutants or stresses have quantitative or non-overlapping annotations (eg. if conditions have doses, or a mutant falls into more than one category simultaneously). We develop a matrix linear model framework that allows us to model relationships between mutants and conditions in a simple, yet flexible multivariate framework. It encodes both categorical and continuous relationships to enhance detection of associations. To handle large datasets, we develop a fast estimation approach that takes advantage of the structure of matrix linear models. We evaluate our method’s performance in simulations and in an E. coli chemical genetic screen, comparing it with an existing univariate approach based on modified t-tests. We show that matrix linear models perform slightly better than the univariate approach when mutants and conditions are classified in non-overlapping categories, and substantially better when conditions can be ordered in dosage categories. Our approach is much faster computationally and is scalable to larger datasets. It is an attractive alternative to current methods, and provides a natural framework extensible to larger, and more complex chemical genetic screens. A Julia implementation of matrix linear models and the code used for the analysis in this paper can be found at https://bitbucket.org/jwliang/mlm_packages and https://bitbucket.org/jwliang/mlm_gs_supplement, respectively.

scholarly journals High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

PLoS Biology ◽  
2016 ◽  
Vol 14 (1) ◽  
pp. e1002341 ◽  
Author(s):  
Alexander J. Meeske ◽  
Christopher D. A. Rodrigues ◽  
Jacqueline Brady ◽  
Hoong Chuin Lim ◽  
Thomas G. Bernhardt ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Throughput ◽  
Genetic Screens ◽  
Sporulation Genes

Chemical Genetics: Drug Screens in Zebrafish

2005 ◽  
Vol 25 (5-6) ◽  
pp. 289-297 ◽  
Author(s):  
Jeroen den Hertog
Keyword(s):  
Biological Activity ◽  
High Throughput ◽  
Large Scale ◽  
Chemical Genetics ◽  
Zebrafish Embryos ◽  
Genetic Screens ◽  
Zebrafish Development ◽  
Chemical Genetic

High throughput chemical genetic screens for compounds with specific biological activity in a whole organism are feasible using zebrafish embryos. At least two medium to large scale drug screens have been carried out to date, leading to the identification of compounds that disturb zebrafish development. Chemical genetics using zebrafish embryos may become an important step in the discovery of drugs and their targets.

scholarly journals Functional genomics in the study of yeast cell polarity: moving in the right direction

2013 ◽  
Vol 368 (1629) ◽  
pp. 20130118 ◽  
Author(s):  
Erin Styles ◽  
Ji-Young Youn ◽  
Mojca Mattiazzi Usaj ◽  
Brenda Andrews
Keyword(s):  
Functional Genomics ◽  
Cell Polarity ◽  
High Throughput ◽  
Budding Yeast ◽  
Model Organism ◽  
Genetic Screens ◽  
High Conservation ◽  
The Right ◽  
Genome Scale ◽  
Integrate Data

The budding yeast Saccharomyces cerevisiae has been used extensively for the study of cell polarity, owing to both its experimental tractability and the high conservation of cell polarity and other basic biological processes among eukaryotes. The budding yeast has also served as a pioneer model organism for virtually all genome-scale approaches, including functional genomics, which aims to define gene function and biological pathways systematically through the analysis of high-throughput experimental data. Here, we outline the contributions of functional genomics and high-throughput methodologies to the study of cell polarity in the budding yeast. We integrate data from published genetic screens that use a variety of functional genomics approaches to query different aspects of polarity. Our integrated dataset is enriched for polarity processes, as well as some processes that are not intrinsically linked to cell polarity, and may provide new areas for future study.

scholarly journals Genome-wide CRISPR-dCas9 screens inE. coliidentify essential genes and phage host factors

2018 ◽  
Author(s):  
François Rousset ◽  
Lun Cui ◽  
Elise Siouve ◽  
Florence Depardieu ◽  
David Bikard
Keyword(s):  
High Throughput ◽  
Essential Genes ◽  
Host Factors ◽  
Null Mutant ◽  
Genetic Screens ◽  
E Coli ◽  
Colanic Acid ◽  
Genome Wide ◽  
A Genome

AbstractHigh-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the chromosome ofE. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome ofE. coli. We then apply the screen to discoverE. coligenes required by phages λ, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by λ enables to identify genes required for the formation of functional λ capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.

scholarly journals Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor

2021 ◽  
Author(s):  
Oded Danziger ◽  
Roosheel S Patel ◽  
Emma J DeGrace ◽  
Mikaela R Rosen ◽  
Brad R Rosenberg
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Life Cycles ◽  
Restriction Factor ◽  
Genetic Screens ◽  
Restriction Factors ◽  
Viral Pathogens ◽  
Interferon Stimulated Genes ◽  
Antiviral Effects ◽  
Crispr Activation

Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.

scholarly journals High-throughput microfluidic cell sorting platform (MICS)

2019 ◽  
Author(s):  
David Philpott ◽  
Peter Aldridge ◽  
Barbara Mair ◽  
Randy Atwal ◽  
Sanna Masud ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Viability ◽  
High Throughput ◽  
Cell Sorting ◽  
Mammalian Cells ◽  
Large Scale ◽  
Genetic Screens ◽  
Large Numbers ◽  
Conventional Cell ◽  
Set Up

Abstract Genome-scale functional genetic screens can be used to interrogate determinants of protein expression modulation of a target of interest. Such phenotypic screening approaches typically require sorting of large numbers of cells (>108). In conventional cell sorting techniques (i.e. fluorescence-activated cell sorting), sorting time, associated with high instrument and operating costs and loss of cell viability, are limiting to the scalability and throughput of these screens. We recently established a rapid and scalable high-throughput microfluidic cell sorting platform (MICS) using immunomagnetic nanoparticles to sort cells in parallel capable of sorting more than 108 HAP1 cells in under one hour while maintaining high levels of cell viability (Ref. 1). This protocol outlines how to set-up MICS for large-scale phenotypic screens in mammalian cells. We anticipate this platform being used for genome-wide functional genetic screens as well as other applications requiring the sorting of large numbers of cells based on protein expression.

scholarly journals Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

2019 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Throughput ◽  
Cell Biology ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

AbstractGenetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

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