scholarly journals Scalable, FACS-Free Genome-Wide Phenotypic Screening

2019 ◽  
Author(s):  
Barbara Mair ◽  
Peter M. Aldridge ◽  
Randy S. Atwal ◽  
Sanna N. Masud ◽  
Meng Zhang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Viability ◽  
High Throughput ◽  
Cell Sorting ◽  
Phenotypic Screening ◽  
Genetic Screens ◽  
Entire Genome ◽  
Genome Wide ◽  
N Terminus ◽  
Genome Scale

ABSTRACTGenome-scale functional genetic screens can identify key regulators of a phenotype of interest, such as determinants of protein expression or modification. Here, we present a rapid, high-throughput approach to phenotypic CRISPR-Cas9 screening. To study factors that modulate the display of CD47 on the cell surface, we processed an entire genome-wide screen containing more than 108cells in under one hour and maintained high levels of cell viability using a highly scalable cell sorting technology. We robustly identified modulators of CD47 function including QPCTL, an enzyme required for formation of the pyroglutamyl modification at the N-terminus of this protein.

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting

2019 ◽  
Vol 3 (10) ◽  
pp. 796-805 ◽  
Author(s):  
Barbara Mair ◽  
Peter M. Aldridge ◽  
Randy S. Atwal ◽  
David Philpott ◽  
Meng Zhang ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
Phenotypic Screening ◽  
Genome Wide

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 Up, down, and out: optimized libraries for CRISPRa, CRISPRi, and CRISPR-knockout genetic screens

2018 ◽  
Author(s):  
Kendall R Sanson ◽  
Ruth E Hanna ◽  
Mudra Hegde ◽  
Katherine F Donovan ◽  
Christine Strand ◽  
...  
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Genes ◽  
Open Reading Frames ◽  
Genetic Screens ◽  
Genome Wide ◽  
Comparable Performance ◽  
Modulation Of Gene Expression ◽  
Genome Scale ◽  
Level Performance

ABSTRACTAdvances in CRISPR-Cas9 technology have enabled the flexible modulation of gene expression at large scale. In particular, the creation of genome-wide libraries for CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) has allowed gene function to be systematically interrogated. Here, we evaluate numerous CRISPRko libraries and show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we developed genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese). Negative selection screens showed that Dolcetto substantially outperforms existing CRISPRi libraries with fewer sgRNAs per gene and achieves comparable performance to CRISPRko in the detection of gold-standard essential genes. We also conducted positive selection CRISPRa screens and show that Calabrese outperforms the SAM library approach at detecting vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.tracr

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.

On-chip cell sorting by high-speed local-flow control using dual membrane pumps

Lab on a Chip ◽  
2017 ◽  
Vol 17 (16) ◽  
pp. 2760-2767 ◽  
Author(s):  
Shinya Sakuma ◽  
Yusuke Kasai ◽  
Takeshi Hayakawa ◽  
Fumihito Arai
Keyword(s):  
Cell Viability ◽  
Success Rate ◽  
High Throughput ◽  
Microfluidic Chip ◽  
Euglena Gracilis ◽  
Cell Sorting ◽  
High Speed ◽  
Local Flow ◽  
On Chip ◽  
Membrane Pumps

We achieved high-throughput on-chip sorting of large cells by using on-chip dual membrane pumps integrated with a high-rigidity microfluidic chip. In the case of Euglena gracilis sorting, the throughput was 23 kHz with 92.8% success rate, 95.8% purity, and 90.8% cell viability.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

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