Inserting dCas9 and single-guide RNAs into Drosophila using molecular cloning methods
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Non-coding DNA in the human genome is widely studied to investigate its effect on coding DNA and gene expression. Non-coding DNA contains cis-regulatory elements that influence transcription of genes upstream, downstream, or nearby. These regulatory elements have largely been studied as enhancers that promote the transcription of genes. To explore these regulatory elements as silencers, we chose validated bifunctional elements to study their silencing capability and their chromatin markers. We used chromatin immunoprecipitation methods with dCas9 to target these elements using single-guide RNAs (sgRNAs). We experimented with various cloning methods to insert dCas9 into the pUAS vector. We initially planned to use the Gibson Assembly method, but after no success, we tried site-directed mutagenesis and traditional cloning with restriction enzymes. We were able to successfully insert dCas9 into the pUAS vector with traditional cloning, and we were then able to inject the construct into Drosophila melanogaster. We designed sgRNAs to target desired elements of DNA that we chose to study as cis-regulatory elements. The sgRNA sequences were cloned into the pCFD5 vector and injected into another line of flies. The transgenic flies containing the pUAS/dCas9 plasmid will then be crossed with the flies containing the pCFD5/sgRNA to develop offspring that express the target elements and could undergo chromatin pulldown to examine the bifunctional regulation of these DNA elements in cells. Results from a quantitative PCR (qPCR) assay on Drosophila expressing the cloned pUAS vector with dCas9 and a sgRNA for the white gene showed chromatin pulldown efficiency and successful transfection. The Drosophila chromatin targeted by the sgRNAs will be pulled down, solubilized, and then analyzed on a western blot to screen for chromatin modifications, primarily histone modifications. We can then identify chromatin markers associated with elements when they act as silencers in the mesoderm versus when they act as non- mesodermal enhancers. We can also determine if the silencer acts by interacting with a promoter or with an enhancer to repress gene expression. If ENCODE can profile the data found in this project, the chromatin markers can act as a predictive tool for the identification of silencers.
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