RT Journal Article
SR Electronic
T1 CRISPRi is not strand-specific and redefines the transcriptional landscape
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 156950
DO 10.1101/156950
A1 Françoise S. Howe
A1 Andrew Russell
A1 Afaf El-Sagheer
A1 Anitha Nair
A1 Tom Brown
A1 Jane Mellor
YR 2017
UL http://biorxiv.org/content/early/2017/06/28/156950.abstract
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.