CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape
- University of Oxford, United Kingdom
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
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.
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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CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape
eLife 6:e29878.
https://doi.org/10.7554/eLife.29878
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