Optical Pooled Screens in Human Cells

David Feldman; Avtar Singh; Jonathan L Schmid-Burgk; Rebecca J Carlson; Anja Mezger; Anthony J Garrity; Feng Zhang; Paul C Blainey

doi:10.1016/j.cell.2019.09.016

Optical Pooled Screens in Human Cells

Cell. 2019 Oct 17;179(3):787-799.e17. doi: 10.1016/j.cell.2019.09.016.

Authors

David Feldman¹, Avtar Singh², Jonathan L Schmid-Burgk², Rebecca J Carlson³, Anja Mezger⁴, Anthony J Garrity², Feng Zhang⁵, Paul C Blainey⁶

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Physics, MIT, Cambridge, MA 02142, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Health Sciences and Technology, MIT, Cambridge, MA 02142, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biological Engineering, MIT, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02142, USA; Department of Brain and Cognitive Science, MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, MIT, Cambridge, MA 02142, USA.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biological Engineering, MIT, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA 02142, USA. Electronic address: pblainey@broadinstitute.org.

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.

Keywords: CRISPR; functional genomics; high-content screening; in situ sequencing; optical pooled screen; pooled screen.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
CRISPR-Cas Systems
Cell Line
Cell Nucleus / genetics
Cell Nucleus / metabolism
Genetic Testing*
Genomics*
Humans
Mediator Complex / genetics
NF-kappa B / genetics*
RNA, Guide, CRISPR-Cas Systems / genetics
Transcription Factor RelA / genetics*

Substances

Mediator Complex
NF-kappa B
RELA protein, human
RNA, Guide, CRISPR-Cas Systems
Transcription Factor RelA

Abstract

Publication types

MeSH terms

Substances

Grants and funding