1. Chromosomes and Gene Expression
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CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape

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Cite as: eLife 2017;6:e29878 doi: 10.7554/eLife.29878

Abstract

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9 binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

Article and author information

Author details

  1. Françoise S Howe

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  2. Andrew Russell

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  3. Anna R Lamstaes

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  4. Afaf El-Sagheer

    Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0001-8706-1292
  5. Anitha Nair

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  6. Tom Brown

    Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0002-6538-3036
  7. Jane Mellor

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    For correspondence
    jane.mellor@bioch.ox.ac.uk
    Competing interests
    Jane Mellor, Holds stock in Oxford BioDynamics Ltd., Chronos Therapeutics Ltd., and Sibelius Ltd. but these holdings present no conflict of interest with work in this article..
    ORCID icon 0000-0002-5196-3734

Funding

Biotechnology and Biological Sciences Research Council (BB/J001694/2)

  • Jane Mellor

Biotechnology and Biological Sciences Research Council (BB/J001694/2)

  • Tom Brown

Wellcome (209897/Z/17/Z)

  • Anna R Lamstaes

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ali Shilatifard, Reviewing Editor, Northwestern University, United States

Publication history

  1. Received: June 23, 2017
  2. Accepted: October 22, 2017
  3. Accepted Manuscript published: October 23, 2017 (version 1)
  4. Version of Record published: November 1, 2017 (version 2)

Copyright

© 2017, Howe et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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