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      Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms

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          Abstract

          The cellular signalling process is a highly complex mechanism, involving multiple players, which together orchestrate the cell’s response to environmental changes and perturbations. Given the multitude of genes that participate in the process of cellular signalling, its study in a genome-wide manner has proven challenging. Recent advances in gene editing technologies, including clustered regularly-interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) approaches, have opened new opportunities to investigate global regulatory signalling programs of cells in an unbiased manner. In this review, we focus on how the application of pooled genetic screening approaches using the CRISPR/Cas9 system has contributed to a systematic understanding of cellular signalling processes in normal and disease contexts.

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          Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

          Hiroko Koike-Yusa, Yilong Li, E-Pien Tan … (2014)
          Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
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            Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

            Julia Joung, Silvana Konermann, Jonathan S. Gootenberg … (2017)
            This protocol from Feng Zhang's lab enables genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system, as sgRNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening.
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              Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

              Andrew Aguirre, Robin Meyers, Barbara Weir … (2016)
              The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: MDPI
                ISSN (Electronic): 1422-0067
                Publication date (Electronic): 21 March 2018
                Publication date Collection: April 2018
                Volume: 19
                Issue: 4
                Electronic Location Identifier: 933
                Affiliations
                [1 ]European Bioinformatics Institute, European Molecular Biology Laboratory, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; petsalaki@ 123456ebi.ac.uk
                [2 ]Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK
                Author notes
                [* ]Correspondence: sumana@ 123456ebi.ac.uk or ss30@ 123456sanger.ac.uk ; Tel.: +44-0122-3492-665
                Author information
                https://orcid.org/0000-0003-0598-2181
                https://orcid.org/0000-0002-8294-2995
                Article
                Publisher ID: ijms-19-00933
                DOI: 10.3390/ijms19040933
                PMC ID: 5979383
                PubMed ID: 29561791
                SO-VID: b4edfc9b-68fe-4739-bfa4-967a813e0bd3
                Copyright © © 2018 by the author.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 26 February 2018
                Date accepted : 18 March 2018
                Categories
                Subject: Review

                ScienceOpen disciplines: Molecular biology
                Keywords: crispr/cas9,genome-wide screens,cellular signalling
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: crispr/cas9, genome-wide screens, cellular signalling

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