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.

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.

scholarly journals Measuring error rates in genomic perturbation screens: gold standards for human functional genomics

2014 ◽  
Author(s):  
Traver Hart ◽  
Kevin R. Brown ◽  
Fabrice Sircoulomb ◽  
Robert Rottapel ◽  
Jason Moffat
Keyword(s):  
Mammalian Cells ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Gene Knockout ◽  
Error Rates ◽  
Measuring Error ◽  
Technological Advancement ◽  
Loss Of Function ◽  
False Discovery Rates ◽  
Gold Standard Reference

AbstractTechnological advancement has opened the door to systematic genetics in mammalian cells. Genome-scale loss-of-function screens can assay fitness defects induced by partial gene knockdown, using RNA interference, or complete gene knockout, using new CRISPR techniques. These screens can reveal the basic blueprint required for cellular proliferation. Moreover, comparing healthy to cancerous tissue can uncover genes that are essential only in the tumor; these genes are targets for the development of specific anticancer therapies. Unfortunately, progress in this field has been hampered by offtarget effects of perturbation reagents and poorly quantified error rates in large-scale screens. To improve the quality of information derived from these screens, and to provide a framework for understanding the capabilities and limitations of CRISPR technology, we derive gold-standard reference sets of essential and nonessential genes, and provide a Bayesian classifier of gene essentiality that outperforms current methods on both RNAi and CRISPR screens. Our results indicate that CRISPR technology is more sensitive than RNAi, and that both techniques have nontrivial false discovery rates that can be mitigated by rigorous analytical methods.

scholarly journals A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia–like syndrome in BXH-2 mice

2005 ◽  
Vol 201 (6) ◽  
pp. 881-890 ◽  
Author(s):  
Karine Turcotte ◽  
Susan Gauthier ◽  
Ashleigh Tuite ◽  
Alaka Mullick ◽  
Danielle Malo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Insertional Mutagenesis ◽  
Consensus Sequence ◽  
Interferon Γ ◽  
Chromosome 8 ◽  
Leukemic Cells ◽  
Interleukin 12 ◽  
Recessive Mutation ◽  
Loss Of Function

BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2–specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence–binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-γ in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.

scholarly journals Human loss-of-function variants suggest that partial LRRK2 inhibition is a safe therapeutic strategy for Parkinson’s disease

2019 ◽  
Author(s):  
Nicola Whiffin ◽  
Irina M. Armean ◽  
Aaron Kleinman ◽  
Jamie L. Marshall ◽  
Eric V. Minikel ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Large Scale ◽  
Therapeutic Strategy ◽  
Kinase Activity ◽  
Model Organisms ◽  
Loss Of Function ◽  
Genomic Databases ◽  
Protein Coding

AbstractHuman genetic variants causing loss-of-function (LoF) of protein-coding genes provide natural in vivo models of gene inactivation, which are powerful indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes1,2. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson’s disease3,4, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. Whilst preclinical studies in model organisms have raised some on-target toxicity concerns5–8, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here we systematically analyse LoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD)9 and over 4 million participants in the 23andMe genotyped dataset, to assess their impact at a molecular and phenotypic level. After thorough variant curation, we identify 1,358 individuals with high-confidence predicted LoF variants in LRRK2, several with experimental validation. We show that heterozygous LoF of LRRK2 reduces LRRK2 protein level by ~50% but is not associated with reduced life expectancy, or with any specific phenotype or disease state. These data suggest that therapeutics that downregulate LRRK2 levels or kinase activity by up to 50% are unlikely to have major on-target safety liabilities. Our results demonstrate the value of large scale genomic databases and phenotyping of human LoF carriers for target validation in drug discovery.

scholarly journals Large-Scale Molecular Characterization of Adeno-Associated Virus Vector Integration in Mouse Liver

2005 ◽  
Vol 79 (6) ◽  
pp. 3606-3614 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Xiaolin Wu ◽  
Sally Fuess ◽  
Theresa A. Storm ◽  
David Munroe ◽  
...  
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Hot Spot ◽  
Disease Genes ◽  
Rrna Gene ◽  
Regulatory Sequences ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Adeno Associated Virus ◽  
Vector Integration

ABSTRACT Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

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 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 Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

2018 ◽  
Author(s):  
Andrea Chai ◽  
Ana M. Mateus ◽  
Fazal Oozeer ◽  
Rita Sousa-Nunes
Keyword(s):  
Large Scale ◽  
Genetic Model ◽  
Model Organisms ◽  
Loss Of Function ◽  
Single Strain ◽  
Genetic Manipulations ◽  
Intrinsic Gene ◽  
Genetic Perturbations ◽  
Neural Tumor ◽  
Tool Set

AbstractStudies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones towards a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.Impact statementA novel genetic strategy for induction of reproducible neural tumors (or any other deleterious phenotype) in a single Drosophila stock (applicable to other organisms).

scholarly journals Spatiotemporally controlled genetic perturbation for efficient large-scale studies of cell non-autonomous effects

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Andrea Chai ◽  
Ana M Mateus ◽  
Fazal Oozeer ◽  
Rita Sousa-Nunes
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetic Model ◽  
Model Organisms ◽  
Loss Of Function ◽  
Single Strain ◽  
Genetic Manipulations ◽  
Intrinsic Gene ◽  
Genetic Perturbations ◽  
Tool Set

Studies in genetic model organisms have revealed much about the development and pathology of complex tissues. Most have focused on cell-intrinsic gene functions and mechanisms. Much less is known about how transformed, or otherwise functionally disrupted, cells interact with healthy ones toward a favorable or pathological outcome. This is largely due to technical limitations. We developed new genetic tools in Drosophila melanogaster that permit efficient multiplexed gain- and loss-of-function genetic perturbations with separable spatial and temporal control. Importantly, our novel tool-set is independent of the commonly used GAL4/UAS system, freeing the latter for additional, non-autonomous, genetic manipulations; and is built into a single strain, allowing one-generation interrogation of non-autonomous effects. Altogether, our design opens up efficient genome-wide screens on any deleterious phenotype, once plasmid or genome engineering is used to place the desired miRNA(s) or ORF(s) into our genotype. Specifically, we developed tools to study extrinsic effects on neural tumor growth but the strategy presented has endless applications within and beyond neurobiology, and in other model organisms.

scholarly journals A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Shen Chi ◽  
Arthur Weiss ◽  
Haopeng Wang
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
T Cell ◽  
Large Scale ◽  
Transcriptional Level ◽  
Loss Of Function ◽  
Genetic Screens ◽  
Endogenous Gene ◽  
Cell Signal ◽  
Cell Signal Transduction

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens.

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