| Autor: |
Carleton JB; Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah., Berrett KC; Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah., Gertz J; Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah; jay.gertz@hci.utah.edu. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of visualized experiments : JoVE [J Vis Exp] 2018 Jun 02 (136). Date of Electronic Publication: 2018 Jun 02. |
| DOI: |
10.3791/57883 |
| Abstrakt: |
Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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