Journal Article

Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells

Viswanatha, Raghuvir, Li, Zhongchi, Perrimon, Norbert, Hu, Yanhui

2018

Overview

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells. Genes are made up of DNA and carry the instructions necessary to build an organism. Humans have over 20,000 genes, while other animals, such as fruit flies, have about 14,000. An ongoing challenge in biology is to identify the role of every gene in the human body. Since most of them are conserved in the fruit fly, this insect is one of the most extensively studied organisms. Scientists often use a technique called CRISPR to edit genes. It enables researchers to modify DNA sequences to selectively alter the purpose of a gene or even turn it off to find out what it does. CRISPR requires a guide molecule (for example, sgRNAs), which leads the system to a particular DNA sequence to start the process. Often, researchers create many sgRNAs and deliver them to a large pool of cells with the help of viruses, so that each cell gets a different sgRNA that mutates a different gene. When the cells are then treated with a specific drug, the composition of the sgRNAs in the pool changes, depending on which genes are needed to withstand the drug, and which genes – when turned-off – create cells that are resistant to the drug. Although thousands of mutant flies have been created to investigate how a deactivated or faulty gene can affect the health and behavior of the fly, we still lack meaningful information on about half of their genes. This is partly because the viruses used to deliver sgRNAs in mammals do not work in fly cells. Here, Viswanatha et al. developed a simple protocol to generate cell pools of CRISPR mutants, which uses a new strategy that uses bacteria to deliver DNA to fly cells. This allowed to identify over 1,000 genes necessary for cells to multiply properly, many of which had not been studied before. The technique was also used in combination with drugs to examine the interactions between genes and drugs – an approach that could be further adapted to examine interactions between genes and nutrients, or between genes. This new approach will open doors to systematically uncover the purpose of every gene in the fly. A better understanding of what genes do could help to identify potential genetic weaknesses in certain types of cancer or other diseases, which may lead to the development of more effective treatments. Moreover, the method is likely to work in other insects, for example, mosquitos, where it may uncover new genes involved in mosquito-borne diseases such as malaria or Zika virus.

• Publisher: eLife Science Publications, Ltd,eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Analysis / Animals / Bacteria / Bacterial genetics / Bar codes / Base sequence / Chromosomes and Gene Expression / Computational and Systems Biology / Computational Biology / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / DNA sequencing / Drosophila / Drosophila - genetics / Drosophila cell lines / Drug Interactions / Efficiency / Fitness / Fitness (Genetics) / fitness essentiality / Fruit flies (Tephritidae) / Gene duplication / Gene Expression Regulation - drug effects / Gene Knockout Techniques / Gene Library / Gene therapy / Genes / Genes, Essential / Genetic aspects / Genetic Fitness / Genetic testing / Genome-Wide Association Study / Genomes / Genomic libraries / Genomics / Growth conditions / Inhibitor drugs / Insects / MAPK / Medical research / Mosquitoes / mTOR / Novels / Ontology / Pharmacogenetics / Phenotype / Physiological aspects / Protein Kinase Inhibitors - pharmacology / Pyridones - pharmacology / Pyrimidinones - pharmacology / Rapamycin / Sirolimus - pharmacology / Tools and Resources / TOR protein