RT Journal Article
SR Electronic
T1 Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 164384
DO 10.1101/164384
A1 Antoine Vigouroux
A1 Enno Oldewurtel
A1 Lun Cui
A1 Sven van Teeffelen
A1 David Bikard
YR 2017
UL http://biorxiv.org/content/early/2017/07/18/164384.abstract
AB Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.