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...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer Nature
2016-08-01
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| Series: | Molecular Systems Biology |
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| Online Access: | https://doi.org/10.15252/msb.20166890 |
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| _version_ | 1849738847590547456 |
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| author | Bianca V Gapp Tomasz Konopka Thomas Penz Vineet Dalal Tilmann Bürckstümmer Christoph Bock Sebastian MB Nijman |
| author_facet | Bianca V Gapp Tomasz Konopka Thomas Penz Vineet Dalal Tilmann Bürckstümmer Christoph Bock Sebastian MB Nijman |
| author_sort | Bianca V Gapp |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-d8ea184279a4405fb6d0eab253cd8844 |
| institution | DOAJ |
| issn | 1744-4292 |
| language | English |
| publishDate | 2016-08-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj-art-d8ea184279a4405fb6d0eab253cd88442025-08-20T03:06:27ZengSpringer NatureMolecular Systems Biology1744-42922016-08-011281910.15252/msb.20166890Parallel reverse genetic screening in mutant human cells using transcriptomicsBianca V Gapp0Tomasz Konopka1Thomas Penz2Vineet Dalal3Tilmann Bürckstümmer4Christoph Bock5Sebastian MB Nijman6Nuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd., University of OxfordNuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd., University of OxfordCeMM Research Center for Molecular Medicine of the Austrian Academy of SciencesNuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd., University of OxfordHorizon GenomicsCeMM Research Center for Molecular Medicine of the Austrian Academy of SciencesNuffield Department of Clinical Medicine, Ludwig Cancer Research Ltd., University of OxfordAbstract 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.https://doi.org/10.15252/msb.20166890kinasesmultiplexed RNA sequencingparallel screeningreverse geneticssystematic phenotyping |
| spellingShingle | Bianca V Gapp Tomasz Konopka Thomas Penz Vineet Dalal Tilmann Bürckstümmer Christoph Bock Sebastian MB Nijman Parallel reverse genetic screening in mutant human cells using transcriptomics Molecular Systems Biology kinases multiplexed RNA sequencing parallel screening reverse genetics systematic phenotyping |
| title | Parallel reverse genetic screening in mutant human cells using transcriptomics |
| title_full | Parallel reverse genetic screening in mutant human cells using transcriptomics |
| title_fullStr | Parallel reverse genetic screening in mutant human cells using transcriptomics |
| title_full_unstemmed | Parallel reverse genetic screening in mutant human cells using transcriptomics |
| title_short | Parallel reverse genetic screening in mutant human cells using transcriptomics |
| title_sort | parallel reverse genetic screening in mutant human cells using transcriptomics |
| topic | kinases multiplexed RNA sequencing parallel screening reverse genetics systematic phenotyping |
| url | https://doi.org/10.15252/msb.20166890 |
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