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Reversible inhibition of specific transcription factor-DNA interactions using CRISPR

Ali Shariati, Antonia Dominguez, Marius Wernig, Lei S. Qi, Jan M. Skotheim
doi: https://doi.org/10.1101/282681
Ali Shariati
Department of Biology, Stanford University, Stanford, CA, 94305, USA
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Antonia Dominguez
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
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Marius Wernig
Department of Pathology, Stem Cell Institute, Stanford, CA 94305, USA
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Lei S. Qi
Department of Bioengineering, Stanford University, Stanford, CA 94305, USADepartment of Chemical and Systems Biology, Stanford Univesity, Stanford, CA 94305, USAStanford ChEM-H, Stanford University, Stanford, CA 94305, USA
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Jan M. Skotheim
Department of Biology, Stanford University, Stanford, CA, 94305, USA
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  • For correspondence: skotheim@stanford.edu
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Abstract

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it has been difficult to assay the function of single transcription factor binding sites within larger transcription networks. Here, we developed such a method by using deactivated Cas9 to disrupt binding to specific sites on the genome. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting deactivated Cas9 to a specific Oct4 binding site in the Nanog promoter blocked Oct4 binding, reduced Nanog expression, and slowed division. Multiple guide RNAs allows simultaneous inhibition of multiple binding sites and conditionally-destabilized dCas9 allows rapid reversibility. The method is a novel high-throughput approach to systematically interrogate cis-regulatory function within complex regulatory networks.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 15, 2018.
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Reversible inhibition of specific transcription factor-DNA interactions using CRISPR
Ali Shariati, Antonia Dominguez, Marius Wernig, Lei S. Qi, Jan M. Skotheim
bioRxiv 282681; doi: https://doi.org/10.1101/282681
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Reversible inhibition of specific transcription factor-DNA interactions using CRISPR
Ali Shariati, Antonia Dominguez, Marius Wernig, Lei S. Qi, Jan M. Skotheim
bioRxiv 282681; doi: https://doi.org/10.1101/282681

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