Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Vigouroux, A.; Oldewurtel, E.; Cui, L.; van Teeffelen, S.; Bikard, D.

doi:10.1101/164384

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bioRxiv · 10.1101/164384

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Vigouroux, A.·Oldewurtel, E.·Cui, L.·van Teeffelen, S.·Bikard, D.

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Abstract

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.

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Vigouroux, A., Oldewurtel, E., Cui, L., van Teeffelen, S., Bikard, D.. 2017-07-18. Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes. https://doi.org/10.1101/164384

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