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 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 Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function

2018 ◽  
Author(s):  
Eric Shifrut ◽  
Julia Carnevale ◽  
Victoria Tobin ◽  
Theodore L. Roth ◽  
Jonathan M. Woo ◽  
...  
Keyword(s):  
T Cells ◽  
Large Scale ◽  
Cell Receptor ◽  
Loss Of Function ◽  
Functional Studies ◽  
Genome Wide ◽  
Human T Cells ◽  
T Cell Receptor Signaling ◽  
Genetic Perturbations ◽  
Signaling Components

SUMMARYHuman T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes validated hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling response to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

scholarly journals Semantic Multi-Classifier Systems Identify Predictive Processes in Heart Failure Models across Species

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 158
Author(s):  
Ludwig Lausser ◽  
Lea Siegle ◽  
Wolfgang Rottbauer ◽  
Derk Frank ◽  
Steffen Just ◽  
...  
Keyword(s):  
Heart Failure ◽  
Regulatory Networks ◽  
Large Scale ◽  
Genetic Model ◽  
Model Organism ◽  
Model Organisms ◽  
Classifier Systems ◽  
Experimental Conditions ◽  
Kegg Pathways ◽  
High Level

Genetic model organisms have the potential of removing blind spots from the underlying gene regulatory networks of human diseases. Allowing analyses under experimental conditions they complement the insights gained from observational data. An inevitable requirement for a successful trans-species transfer is an abstract but precise high-level characterization of experimental findings. In this work, we provide a large-scale analysis of seven weak contractility/heart failure genotypes of the model organism zebrafish which all share a weak contractility phenotype. In supervised classification experiments, we screen for discriminative patterns that distinguish between observable phenotypes (homozygous mutant individuals) as well as wild-type (homozygous wild-types) and carriers (heterozygous individuals). As the method of choice we use semantic multi-classifier systems, a knowledge-based approach which constructs hypotheses from a predefined vocabulary of high-level terms (e.g., Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways or Gene Ontology (GO) terms). Evaluating these models leads to a compact description of the underlying processes and guides the screening for new molecular markers of heart failure. Furthermore, we were able to independently corroborate the identified processes in Wistar rats.

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.

Suppressors of a Cold-Sensitive Mutation in Yeast U4 RNA Define Five Domains in the Splicing Factor Prp8 That Influence Spliceosome Activation

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1667-1682 ◽  
Author(s):  
Andreas N Kuhn ◽  
David A Brow
Keyword(s):  
Large Scale ◽  
Splicing Factor ◽  
Cold Sensitivity ◽  
Splicing Factors ◽  
Loss Of Function ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Cold Sensitive ◽  
Spliceosome Activation ◽  
U4 Rna

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

scholarly journals Insights into mammalian biology from the wild house mouse Mus musculus

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Megan Phifer-Rixey ◽  
Michael W Nachman
Keyword(s):  
House Mouse ◽  
Mus Musculus ◽  
Genetic Model ◽  
Inbred Strains ◽  
Mendelian Inheritance ◽  
Model Organisms ◽  
House Mice ◽  
Small Subset ◽  
Wild Mice ◽  
Wide Range

The house mouse, Mus musculus, was established in the early 1900s as one of the first genetic model organisms owing to its short generation time, comparatively large litters, ease of husbandry, and visible phenotypic variants. For these reasons and because they are mammals, house mice are well suited to serve as models for human phenotypes and disease. House mice in the wild consist of at least three distinct subspecies and harbor extensive genetic and phenotypic variation both within and between these subspecies. Wild mice have been used to study a wide range of biological processes, including immunity, cancer, male sterility, adaptive evolution, and non-Mendelian inheritance. Despite the extensive variation that exists among wild mice, classical laboratory strains are derived from a limited set of founders and thus contain only a small subset of this variation. Continued efforts to study wild house mice and to create new inbred strains from wild populations have the potential to strengthen house mice as a model system.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot

2021 ◽  
Author(s):  
Doris Škorić-Milosavljević ◽  
Najim Lahrouchi ◽  
Fernanda M. Bosada ◽  
Gregor Dombrowsky ◽  
Simon G. Williams ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Tetralogy Of Fallot ◽  
Large Scale ◽  
Rare Variants ◽  
Growth Factor Receptor ◽  
Vegf Receptor ◽  
Loss Of Function ◽  
Endothelial Growth Factor

Abstract Purpose Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. Methods We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. Results Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). Conclusion Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

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.

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