PT  - JOURNAL ARTICLE
AU  - Françoise S. Howe
AU  - Andrew Russell
AU  - Afaf El-Sagheer
AU  - Anitha Nair
AU  - Tom Brown
AU  - Jane Mellor
TI  - CRISPRi is not strand-specific and redefines the transcriptional landscape
AID  - 10.1101/156950
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 156950
4099  - http://biorxiv.org/content/early/2017/06/28/156950.short
4100  - http://biorxiv.org/content/early/2017/06/28/156950.full
AB  - 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, binding of sgRNA/dCas9 creates an environment that is permissive for transcription initiation and termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand 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.