title: Parallel reverse genetic screening in mutant human cells using transcriptomics.
abstract: Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease-relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large-scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.
authors.affiliation: Nuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd. University of Oxford, Oxford, UK.
authors.forename: Bianca V
authors.initials: BV
authors.lastname: Gapp
authors.affiliation: Nuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd. University of Oxford, Oxford, UK.
authors.forename: Tomasz
authors.identifier: 0000-0003-3042-4712
authors.initials: T
authors.lastname: Konopka
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Penz
authors.affiliation: Nuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd. University of Oxford, Oxford, UK.
authors.forename: Vineet
authors.initials: V
authors.lastname: Dalal
authors.affiliation: Horizon Genomics, Vienna, Austria.
authors.forename: Tilmann
authors.initials: T
authors.lastname: Bürckstümmer
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Max Planck Institute for Informatics, Saarbrücken, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Bock
authors.affiliation: Nuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd. University of Oxford, Oxford, UK CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Nuffield Department of Clinical Medicine, Target Discovery Institute University of Oxford, Oxford, UK Sebastian.nijman@ludwig.ox.ac.uk.
authors.forename: Sebastian Mb
authors.identifier: 0000-0003-0464-6661
authors.initials: SM
authors.lastname: Nijman
mesh_terms: D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods*; D055786:Gene Knockout Techniques; D005838:Genotype; D006801:Humans; D058977:Molecular Sequence Annotation; D010641:Phenotype; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D011505:Protein-Tyrosine Kinases
keywords: kinases; multiplexed RNA sequencing; parallel screening; reverse genetics; systematic phenotyping