Poster abstracts
Poster number 119 submitted by Victoria Rai
Transcription Regulation using CRISPR/Cas9
Victoria Rai (University of Michigan Department of Chemistry), Julia R. Widom (University of Michigan Department of Chemistry), Nils G. Walter (University of Michigan Department of Chemistry)
Abstract:
Our ability to control site-specific gene expression has been expanded by the gene-editing tool CRISPR/Cas9. With immunity and inherent memory capabilities, this system applies to our investigation in understanding the mechanisms of the RNA polymerase (RNAP) complex in bacteria. More specifically, by regulating the movement of RNAP in T7 phages at a particular site of transcription using CRISPR/Cas9, we are able to block and unblock the transcription process altogether. As transcription errors can result in numerous genetic disorders, like fibrotic conditions and cancers, the ability to “traffic” RNAP is crucial. Through the creation of multiple guide RNAs, each of which contain a specific sequence that directs protein Cas9 to a target site, we have demonstrated the ability to block transcription. More recently, the process has been successfully unblocked, though further investigation in necessary. While transcription assays revealed that transcription is restarted upon unblocking, further suggesting that Cas9 is fully removed, the unblocked RNAP does not fully produce its runoff product in proportion to its stalled product. The aim remains to allow already bound RNAP to resume transcription upon unblocking. Using a number of avenues, the investigation has turned to techniques in lowering RNAP to DNA ratios to avoiding “bumping” of the polymerases and utilizing other polymerases including those from E. coli, and the bacteriophages T7, SP6, and T3. In line with the aims of this investigation, understanding the interactions between these RNAPs and the protein Cas9 on DNA pertains to transcription regulation, allowing for further application of these regulation techniques.
Keywords: CRISPRCas9, Transcription, RNAP