scholarly journals Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

2019 ◽  
Author(s):  
Arshad H. Khan ◽  
Andy Lin ◽  
Richard T. Wang ◽  
Joshua S. Bloom ◽  
Kenneth Lange ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Single Gene ◽  
Pooled Analysis ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Gene Copies ◽  
Radiation Hybrids ◽  
Genome Wide ◽  
Low Pass ◽  
Mammalian Genetic

AbstractGenetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ~2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ~30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

scholarly journals Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier

2020 ◽  
Author(s):  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Dynamic Range ◽  
Quality Data ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Gene Essentiality ◽  
Guide Rna ◽  
Genome Wide ◽  
Improved Model ◽  
Target Effects

AbstractIdentifying essential genes in genome-wide loss of function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome wide genetic screens. Here, we introduce an updated version, BAGEL2, which employs an improved model that offers greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes, and a multi-target correction that reduces false positives from off-target CRISPR guide RNA. We also suggest a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower-quality data in substantially different ways. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

Abstract 403: CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens

2017 ◽  
Author(s):  
Donato Tedesco ◽  
Paul Diehl ◽  
Mikhail Makhanov ◽  
Sylvain Baron ◽  
Alex Chenchik
Keyword(s):  
Loss Of Function ◽  
Genetic Screens ◽  
Gain Of Function ◽  
Genome Wide

scholarly journals A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

2019 ◽  
Author(s):  
Charlotte R. Feddersen ◽  
Lexy S. Wadsworth ◽  
Eliot Y. Zhu ◽  
Hayley R. Vaughn ◽  
Andrew P. Voigt ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Sequence Data ◽  
Sleeping Beauty ◽  
Transposon Mutagenesis ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Sleeping Beauty Transposon ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
The Impact

AbstractThe introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the Sleeping Beauty transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

scholarly journals Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

2020 ◽  
Vol 30 (10) ◽  
pp. 1458-1467
Author(s):  
Arshad H. Khan ◽  
Andy Lin ◽  
Richard T. Wang ◽  
Joshua S. Bloom ◽  
Kenneth Lange ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Pooled Analysis ◽  
Drug Action ◽  
Radiation Hybrids

scholarly journals Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Dynamic Range ◽  
Quality Data ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Gene Essentiality ◽  
Guide Rna ◽  
Genome Wide ◽  
And Performance ◽  
Improved Model ◽  
Sensitivity Specificity

Abstract Background Identifying essential genes in genome-wide loss-of-function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome-wide genetic screens. Results We introduce an updated version, BAGEL2, which employs an improved model that offers a greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes; a multi-target correction that reduces false positives from off-target CRISPR guide RNA; and the implementation of a cross-validation strategy that improves performance ~ 10× over the prior bootstrap resampling approach. We also describe a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower quality data in substantially different ways. Conclusions BAGEL2 substantially improves the sensitivity, specificity, and performance over BAGEL and establishes the new state of the art in the analysis of CRISPR knockout fitness screens. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

scholarly journals Elucidating drug targets and mechanisms of action by genetic screens in mammalian cells

2017 ◽  
Vol 53 (53) ◽  
pp. 7162-7167 ◽  
Author(s):  
Martin Kampmann
Keyword(s):  
Mammalian Cells ◽  
Drug Target ◽  
Drug Targets ◽  
Drug Response ◽  
Target Identification ◽  
Mechanisms Of Action ◽  
Genetic Screens ◽  
Crispr Interference ◽  
Genome Wide ◽  
Drug Target Identification

Genome-wide CRISPR interference (CRISPRi) screens in mammalian cells enable drug target identification and uncover genes controlling drug response.

Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

Science ◽  
2009 ◽  
Vol 326 (5957) ◽  
pp. 1231-1235 ◽  
Author(s):  
Jan E. Carette ◽  
Carla P. Guimaraes ◽  
Malini Varadarajan ◽  
Annie S. Park ◽  
Irene Wuethrich ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Screening Method ◽  
Chromosome 8 ◽  
Host Factors ◽  
Null Alleles ◽  
Model Organisms ◽  
Loss Of Function ◽  
Genetic Screens

Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.

scholarly journals Model-guided design of mammalian genetic programs

2021 ◽  
Vol 7 (8) ◽  
pp. eabe9375
Author(s):  
J. J. Muldoon ◽  
V. Kandula ◽  
M. Hong ◽  
P. S. Donahue ◽  
J. D. Boucher ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Computational Models ◽  
Biological Complexity ◽  
Genetic Circuits ◽  
Cellular Functions ◽  
High Performing ◽  
Design Objective ◽  
Complex Functions ◽  
Mammalian Genetic ◽  
Analog Processing

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

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