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

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 the 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.

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

“…Over the last 3 years, CRISPR-Cas9 has been applied to mammalian screens, including screens to identify mechanisms of resistance or enhanced sensitivity to drugs (9,13), to identify mediators of immune response (14), and to identify enhancer elements (15). To date, however, this approach has not been systematically Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy.…”

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

“…7). Whereas primary dendritic cells of these mice have been successfully used for functional screening in vitro (6,8), their lymphocytes express only low levels of GFP, and their use in CRISPR-mediated screens has not been reported so far. In addition, these mice were not generated on a pure C57BL/6 background.…”

“…As we were interested in small-scale screening, a key consideration was to use only few, but reliable single guide RNAs (sgRNAs) without the necessity of sgRNA-testing experiments before screening. Previous studies have shown large variations in sgRNA efficiencies (8,(10)(11)(12). Multiple sgRNA design programs have been developed, but none of them combines all of the criteria that we developed in laboratory to reach optimal sgRNA activity and specificity, including on-target efficiency, genome-wide off-target predictions taking into account the impact of mismatch type and position, targeting of all isoforms of genes, and conservation of functional secondary structure of the sgRNA (13).…”

Genome-Editing Technologies: Principles and Applications

“…CRISPR has also facilitated multiplexed metabolic pathway engineering in yeast at high efficiencies (Jakociunas et al 2015a,b), as well as random mutagenesis of yeast chromosomal DNA for phenotypic screening of desired mutants (Ryan et al 2014). Indeed, genome-wide CRISPR-based knockout screens hold tremendous potential for functional genomics (Hilton and Gersbach 2015), having facilitated the discovery of genomic loci that confer drug resistance to cells (Koike-Yusa et al 2014;Shalem et al 2014;Wang et al 2014a;Zhou et al 2014), uncovered how cells can control induction of the host immune response (Parnas et al 2015), provided new insights into the genetic basis of cellular fitness (Hart et al 2015;Wang et al 2015b), and even shed light on how certain viruses induce cell death . Genome-wide CRISPR screens can also facilitate the discovery of functional noncoding elements (Kim et al 2013b;Korkmaz et al 2016), and provide a means for studying the structure and evolution of the human genome.…”