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Elife. 2017 Oct 23;6. pii: e29878. doi: 10.7554/eLife.29878.

CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape.

Author information

1
Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
2
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom.
3
Chemistry Branch, Faculty of Petroleum and Mining Engineering, Suez University, Suez, Egypt.

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.

KEYWORDS:

CRISPRi; HMS2 and GAL1; S. cerevisiae; chromosomes; genes; strand-specificity; transcription initiation; transcription roadblock; transcription termination

PMID:
29058669
PMCID:
PMC5665645
DOI:
10.7554/eLife.29878
[Indexed for MEDLINE]
Free PMC Article

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