Optical Pooled Screens in Human Cells
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic scre...
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Published in | Cell Vol. 179; no. 3; pp. 787 - 799.e17 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
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United States
Elsevier Inc
17.10.2019
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Subjects | |
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Abstract | Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
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•In situ sequencing of perturbations or barcodes enables image-based pooled screens•p65 translocation is assayed by imaging in fixed and live cell pools•Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-κB
A screening approach that combines high-content imaging with in situ sequencing can identify genes that affect spatially and temporally defined phenotypes like morphology and subcellular localization, expanding the list of scientific questions that can be asked with genetic tools. |
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AbstractList | Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability, but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted
in situ
sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in NF-κB signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
A screening approach that combines high-content imaging with in situ sequencing can identify genes that affect spatially and temporally defined phenotypes like morphology and subcellular localization, expanding the list of scientific questions that can be asked with CRISPR-based tools. Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries. [Display omitted] •In situ sequencing of perturbations or barcodes enables image-based pooled screens•p65 translocation is assayed by imaging in fixed and live cell pools•Pooled live-cell screen identifies MED12 and MED24 as negative regulators of NF-κB A screening approach that combines high-content imaging with in situ sequencing can identify genes that affect spatially and temporally defined phenotypes like morphology and subcellular localization, expanding the list of scientific questions that can be asked with genetic tools. Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries. |
Author | Feldman, David Singh, Avtar Blainey, Paul C. Schmid-Burgk, Jonathan L. Carlson, Rebecca J. Garrity, Anthony J. Mezger, Anja Zhang, Feng |
AuthorAffiliation | 1 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142, USA 5 Department of Biological Engineering, MIT, Cambridge, Massachusetts, 02142, USA 10 These authors contributed equally to this work 9 Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, 02142, USA 6 McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts, 02142, USA 3 Department of Health Sciences and Technology, MIT, Cambridge, Massachusetts, 02142, USA 8 Howard Hughes Medical Institute, MIT, Cambridge, Massachusetts, 02142, USA 4 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden 11 Lead contact 2 Department of Physics, MIT, Cambridge, Massachusetts, 02142, USA 7 Department of Brain and Cognitive Science, MIT, Cambridge, Massachusetts, 02142, USA |
AuthorAffiliation_xml | – name: 3 Department of Health Sciences and Technology, MIT, Cambridge, Massachusetts, 02142, USA – name: 11 Lead contact – name: 1 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, 02142, USA – name: 5 Department of Biological Engineering, MIT, Cambridge, Massachusetts, 02142, USA – name: 10 These authors contributed equally to this work – name: 4 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden – name: 8 Howard Hughes Medical Institute, MIT, Cambridge, Massachusetts, 02142, USA – name: 9 Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, 02142, USA – name: 7 Department of Brain and Cognitive Science, MIT, Cambridge, Massachusetts, 02142, USA – name: 2 Department of Physics, MIT, Cambridge, Massachusetts, 02142, USA – name: 6 McGovern Institute for Brain Research at MIT, Cambridge, Massachusetts, 02142, USA |
Author_xml | – sequence: 1 givenname: David surname: Feldman fullname: Feldman, David organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 2 givenname: Avtar surname: Singh fullname: Singh, Avtar organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 3 givenname: Jonathan L. surname: Schmid-Burgk fullname: Schmid-Burgk, Jonathan L. organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 4 givenname: Rebecca J. surname: Carlson fullname: Carlson, Rebecca J. organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 5 givenname: Anja surname: Mezger fullname: Mezger, Anja organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 6 givenname: Anthony J. surname: Garrity fullname: Garrity, Anthony J. organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 7 givenname: Feng surname: Zhang fullname: Zhang, Feng organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA – sequence: 8 givenname: Paul C. surname: Blainey fullname: Blainey, Paul C. email: pblainey@broadinstitute.org organization: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/31626775$$D View this record in MEDLINE/PubMed http://kipublications.ki.se/Default.aspx?queryparsed=id:142117530$$DView record from Swedish Publication Index |
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Keywords | pooled screen CRISPR optical pooled screen in situ sequencing functional genomics high-content screening |
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Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 D.F. designed the approach with input from all authors. D.F., A.S., A.M., A.J.G., R.J.C. and J.S.B performed experiments. D.F. analyzed data. J.S.B and D.F. designed the NF-kB screen. P.C.B. and F.Z. supervised the research. D.F., A.S., J.S.B, and P.C.B. wrote the manuscript with contributions from all authors. AUTHOR CONTRIBUTIONS |
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SubjectTerms | Animals Cell Line Cell Nucleus - genetics Cell Nucleus - metabolism CRISPR CRISPR-Cas Systems functional genomics Genetic Testing Genomics high-content screening Humans in situ sequencing Mediator Complex - genetics Medicin och hälsovetenskap NF-kappa B - genetics optical pooled screen pooled screen RNA, Guide, CRISPR-Cas Systems - genetics Transcription Factor RelA - genetics |
Title | Optical Pooled Screens in Human Cells |
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