Team:Waterloo/InDepthDesign/Crispr
CRISPR
CRISPR (clustered regularly interspaced short palindromic repeats) is a system used for targeting specific sites of a genome. The system is comprised of a Cas protein and an sgRNA which is a short guide RNA, usually about 20nt, which specifies the target site on the genome. CRISPR is a very popular tool in genome editing because of its ability to cut specific sites, however it was originally discovered as an adaptive immune system in bacteria. This function of the CRISPR system allows for a targeted knockout of a protein in question.
Our system allows for an automatic upregulation of a protein which enables researchers to see if a protein can cure a Psi+ response. It would also be useful to be able to see if supressing the expression of a protein can cure the Psi+ response. For instance, upregulating HSP104 can cure the response by refolding misfolded SUP35 but downregulating HSP104 can also cure the response because the SUP35 aggregates need HSP104 in order to replicate. Adding a CRISPR system behind a stop codon, which targets a protein can cause downregulation of that protein during a Psi+ state.
Most CRISPR systems use a Cas protein called Cas9 which makes a cut after binding to the DNA. Our system would use dCas9 which is a Cas protein that binds to a site and prevents transcription of that protein. This acts as a more reliable method for knocking out a protein, since cuts can sometimes be repaired effectively through non-homologous end joining.
All sgRNAs must start with a certain sequence of nucleotides called a PAM site. This is so that the Cas protein can bind to the DNA before reading the specific sequence. The most common PAM site is NGG... something something, yeast.
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- Mumberg, D., Müller, R., and Funk, M. (1994). Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Research, 22:25. 5767-5768.
