A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks

Oren Parnas; Marko Jovanovic; Thomas M Eisenhaure; Rebecca H Herbst; Atray Dixit; Chun Jimmie Ye; Dariusz Przybylski; Randall J Platt; Itay Tirosh; Neville E Sanjana; Ophir Shalem; Rahul Satija; Raktima Raychowdhury; Philipp Mertins; Steven A Carr; Feng Zhang; Nir Hacohen; Aviv Regev

doi:10.1016/j.cell.2015.06.059

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks

Cell. 2015 Jul 30;162(3):675-86. doi: 10.1016/j.cell.2015.06.059. Epub 2015 Jul 16.

Authors

Oren Parnas¹, Marko Jovanovic¹, Thomas M Eisenhaure², Rebecca H Herbst³, Atray Dixit⁴, Chun Jimmie Ye⁵, Dariusz Przybylski¹, Randall J Platt⁶, Itay Tirosh¹, Neville E Sanjana⁷, Ophir Shalem⁸, Rahul Satija⁹, Raktima Raychowdhury¹, Philipp Mertins¹, Steven A Carr¹, Feng Zhang¹⁰, Nir Hacohen¹¹, Aviv Regev¹²

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA.
⁵ Institute for Human Genetics, Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁷ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁹ New York Genome Center, New York, NY 10013, USA; New York University, Center for Genomics and Systems Biology, New York, NY 10012, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA; Department of Medicine, Harvard Medical School, Boston MA 02114. Electronic address: nhacohen@broadinstitute.org.
¹² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02140, USA. Electronic address: aregev@broadinstitute.org.

Abstract

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Bone Marrow Cells / immunology
CRISPR-Cas Systems*
Cell Differentiation
Cell Survival
Dendritic Cells / cytology
Dendritic Cells / immunology
Gene Knockout Techniques
Gene Regulatory Networks
Genetic Techniques*
Hexosyltransferases / metabolism
Immunity, Innate*
Membrane Proteins / metabolism
Mice
Mice, Transgenic
Toll-Like Receptor 4 / immunology
Tumor Necrosis Factor-alpha / immunology

Substances

Membrane Proteins
Tlr4 protein, mouse
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha
Hexosyltransferases
dolichyl-diphosphooligosaccharide - protein glycotransferase

Associated data

GEO/GSE67164

Abstract

Publication types

MeSH terms

Substances

Associated data

Grants and funding