Difference between revisions of "Team:Waterloo/InDepthDesign/Crispr"
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<p>Figure 1: CRISPR action, demonstrating how Cas9 binds to sgRNA to cleave target DNA.</p> | <p>Figure 1: CRISPR action, demonstrating how Cas9 binds to sgRNA to cleave target DNA.</p> | ||
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| − | <p>Our system allows | + | <p>Our system is a tool that allows the overexpression a protein of interest, which could be used to determine if the protein can slow the [PSI+] response or even cure it. It would also be useful to see if the opposite (a decrease in the expression of a protein of interest) could have similar effects. For instance, overexpressing hsp104 can cure the response by breaking down and refolding misfolded sup35, but knocking down hsp104 expression can also cure the response because the sup35 aggregates need hsp104 in order to replicate. The addition of a CRISPR interference system using dCas9 allows the targeting a protein of interest to cause this knockdown of expression. If we use our system to insert a premature stop codon in front of the CRISPR gene, then it will only be expressed during a [PSI+] state.</p> |
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Revision as of 02:32, 20 October 2016
CRISPR
Figure 1: CRISPR action, demonstrating how Cas9 binds to sgRNA to cleave target DNA.
CRISPR (clustered regularly interspaced short palindromic repeats) is a system used for targeting specific sites of a genome (Cong, 2013; Jinek, 2012). 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(Cong, 2013; Jinek, 2012). 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 (Doudna, 2014). This function of the CRISPR system allows for a targeted knockout of a protein in question.
Our system is a tool that allows the overexpression a protein of interest, which could be used to determine if the protein can slow the [PSI+] response or even cure it. It would also be useful to see if the opposite (a decrease in the expression of a protein of interest) could have similar effects. For instance, overexpressing hsp104 can cure the response by breaking down and refolding misfolded sup35, but knocking down hsp104 expression can also cure the response because the sup35 aggregates need hsp104 in order to replicate. The addition of a CRISPR interference system using dCas9 allows the targeting a protein of interest to cause this knockdown of expression. If we use our system to insert a premature stop codon in front of the CRISPR gene, then it will only be expressed during a [PSI+] state.
Figure 2: CRISPRi knockdown action, demonstrating how dCas9 binds to sgRNA to prevent expression.
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.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., & Zhang, F., 2013. Multiplex genome engineering using CRISPR/Cas systems. Science Feb 15;339(6121):819-23.
Doudna, J.A. & Charpentier, E., 2014. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), pp.1258096–1258096.
Jinek, M. et al., 2012. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), p.816 LP-821.
