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Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds.

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      Abstract

      Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.

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      Affiliations
      [1 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA.
      [2 ] Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Energy Biosciences Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
      [3 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
      [4 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA.
      [5 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, CA 94158, USA.
      [6 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA.
      [7 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA. Electronic address: slqi@stanford.edu.
      [8 ] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA. Electronic address: wendell.lim@ucsf.edu.
      Journal
      Cell
      Cell
      1097-4172
      0092-8674
      Jan 15 2015
      : 160
      : 1-2
      S0092-8674(14)01570-0
      10.1016/j.cell.2014.11.052
      25533786
      4297522
      NIHMS645765
      Copyright © 2015 Elsevier Inc. All rights reserved.

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