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      Cornerstones of CRISPR–Cas in drug discovery and therapy

      Author(s): , , , ,
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      Journal: Nature Reviews Drug Discovery
      Publisher: Springer Nature

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          Abstract

          The use of CRISPR–Cas technology for gene editing has rapidly become widespread. Here, Corn and colleagues discuss the applications of this revolutionary tool in drug discovery and development, describing how it could make substantial contributions to target identification and validation, animal models and cell-based therapies.

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          Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

          Isaac B. Hilton, Anthony M. D’Ippolito, Christopher M. Vockley … (2015)
          Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.
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            Central dogma of molecular biology.

            FRANCIS CRICK (1970)
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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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                Author and article information

                Journal
                Title: Nature Reviews Drug Discovery
                Abbreviated Title: Nat Rev Drug Discov
                Publisher: Springer Nature
                ISSN (Print): 1474-1776
                ISSN (Electronic): 1474-1784
                Publication date Created: December 23 2016
                Publication date (Print): December 23 2016
                Volume:
                Issue:
                Article
                DOI: 10.1038/nrd.2016.238
                PMC ID: 5459481
                PubMed ID: 28008168
                SO-VID: 5dc68343-d471-4051-b89c-a1d14515288a
                Copyright © © 2016
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