Difference between revisions of "Team:Waterloo/InDepthDesign/Crispr"

(Yeast paragraph, more references)
 
(12 intermediate revisions by 4 users not shown)
Line 1: Line 1:
 
{{Template:Waterloo}}
 
{{Template:Waterloo}}
 
<html>
 
<html>
 +
<div class="labsticker">
 +
  Lab and Design
 +
</div>
 +
<div class="mathsticker">
 +
  Math Modelling
 +
</div>
 
<body>  
 
<body>  
  
 
<div class="container-fluid">
 
<div class="container-fluid">
 
   <div class="bloodorangeisacolour">
 
   <div class="bloodorangeisacolour">
−
     <div class="header-image">
+
     <div class="header-image" style="background-image: url(https://static.igem.org/mediawiki/2016/6/6c/T--Waterloo--office2.jpg);">
−
       <h3 class="header-text"><span style="color: white; background-color: #000;opacity: 0.8;">CRISPR</span></h3>
+
       <h3 class="header-text"><span style="color: white; background-color: #000;opacity: 0.8;">CRISPRi</span></h3>
 
     </div>
 
     </div>
 
   </div>
 
   </div>
Line 12: Line 18:
  
 
<div class="wcontent-box">
 
<div class="wcontent-box">
−
<div class="wcontent-title">
+
  <div class="wcontent-title">
−
Summary
+
    Summary
 +
  </div>
 +
  <div class="wcontent">
 +
  <img class="wcontent-img-solo37" src="https://static.igem.org/mediawiki/2016/6/67/Crispr-cas9.png"/>
 +
    <div class="wcontent-caption">
 +
      <p>Figure 1: CRISPR action, demonstrating how Cas9 binds to sgRNA to cleave target DNA.</p>
 +
    </div>
 +
    <p>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.
 +
    </p>
 +
  </div>
 
</div>
 
</div>
  
−
<div class="wcontent">
+
<div class="wcontent-box">
−
 
+
  <div class="wcontent-title">
−
<p>
+
    Application for PriON to PriOFF
−
It was necessary for us to choose promoters that expressed the constructs we were cloning into yeast cells. We wanted to characterize promoter expression with GFP before we decided which promoter to use in our system with the CFP-Hsp104 construct. Furthermore, we needed to induce a [PSI+] state by overexpression of Sup35 in the cell by inserting another copy of Sup35 into the system via a plasmid. Overall, we had two plasmids that we wanted to easily induce at any time.
+
  </div>
−
</p>
+
  <div class="wcontent">
−
 
+
    <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>
−
<p>
+
  </div>
−
We chose to characterize four main promoters that are commonly found in yeast: Gal1, Adh1, Met25, and Cup1. We cloned all of these into the pXP218 plasmid and then transformed them into yeast to test their strength with and without the presence of inducers. All of the promoters we picked are inducible except for Adh1, which acts constitutively. We made the promoter constructs by the traditional restriction digest cloning method after order the synthesized promoters with a GFP fluorescent marker and doing PCR amplification with appropriate primers.  
+
−
</p>
+
−
 
+
−
<p>
+
−
Specifically, The promoters were added into the pXP218 plasmid between the restriction enzymes SphI and SalI, which are near the multiple cloning site in the plasmid.The criteria required for the plasmid were the following: there are no T7 promoter regions near the promoter insertion site, so that T7 does not give a false positive by increasing expression of the promoter being tested, it had to be able to replicate in yeast, be able to be constructed in Escherichia coli,  have a CEN/ARS or a 2 micron origin of replication, and have a bacterial origin of replication. Having a selective marker in the plasmid was also important an important factor so that we were able to select for transformants into yeast; some markers include LEU2, URA3, HIS3.
+
−
 
+
−
<img class="wcontent-img-solo37" src="https://static.igem.org/mediawiki/2016/b/b3/Features_of_pXP218.png"/>
+
−
 
+
−
<div class="wcontent-caption">
+
−
<p> Pxp218 yeast expression vector showing the main features which include: the backbone of pUC18, a vector size of 6226, URA3 selectable marker, ampicillin bacterial resistance, temperature growth at 37 °C, with growth stains DH5alpha and a high copy number. </p>
+
−
 
+
−
</div>
+
−
</div>
+
 
</div>
 
</div>
  
 
<div class="wcontent-box">
 
<div class="wcontent-box">
−
<div class="wcontent-title">
+
  <div class="wcontent-title">
−
Why we chose our restriction enzymes
+
    dCas9
−
</div>
+
  </div>
−
<div class="wcontent">
+
  <div class="wcontent">
−
<p>
+
  <img class="wcontent-img-solo37" src="https://static.igem.org/mediawiki/2016/f/ff/Igem_crispri.png"/>
−
When selecting the section of the plasmid to insert the Gal1 promoter and GFP, it needed to be ensured that the restriction enzymes chosen for the plasmid were not located within the Gal1/GFP sequence. This process for ensuring that the selected enzyme was not in our insert was repeated for each promoter with the fused GFP. Furthermore, restriction enzymes that would be used to digest our vector backbone would need to be found at the ends of the insert. This could be done through PCR.The order for each construct was the promoter itself to initiate transcription, the kozak sequence to start translation, the stop codon to stop translation, GFP, ORF - a terminator to stop transcription, and two restriction enzyme sites to allow the vector to be cloned.To design the promoters, research was conducted regarding how much space would be required between the kozak sequence and each promoter as well as how much space is needed between all the elements. Our team used other plasmids with the same promoters we are testing and GFP as a template when designing the sequence between the promoter and GFP in our promoter to ensure the correct sequences were used. Each promoter insert that has been through PCR was ligated into the pSB1C3 and pXP218 plasmids. To ligate into the pSB1C3 plasmid specifically, we cut with E and P ligating into pXP218, and used SphI and SalI to cut. These restriction sites were created at the ends of our inserts by using primers that had the correct site.
+
    <div class="wcontent-caption">
−
 
+
      <p>Figure 2: CRISPRi knockdown action, demonstrating how dCas9 binds to sgRNA to prevent expression.</p>
−
</p>
+
    </div>
−
<p>
+
    <p>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. </p>
−
For Gal1, the SphI and SalI restriction sites were used to create sticky ends when digested. This would allow the insert to properly ligate in the correct orientation into the PXP218 plasmid. GFP is required to be able to use fluorimetry on the plasmids to analyze fluorescence and to test our promotor. A small set of twelve nitrogenous bases between the GFP and Gal1 that was taken from other plasmids between a Gal1 and a various feature. Our team researched various plasmids containing Gal1 and took a sequence directly after the Gal1 promoter. The CYC1 terminator is used to end the insert while there is a biobrick prefix directly after the terminator. This is done to ensure it can be tested as an iGEM BioBrick. For Cup1 and Adh1, the same parts were used, except the actual promoter changed.
+
  </div>
−
</p>
+
−
</div>
+
 
</div>
 
</div>
  
 
<div class="wcontent-box">
 
<div class="wcontent-box">
−
<div class="wcontent-title">
+
  <div class="wcontent-title">
−
Our Promoters in Nature
+
    sgRNAs
−
</div>
+
  </div>
−
<div class="wcontent">
+
  <div class="wcontent">
−
<p>
+
    <p>All sgRNAs must start with a certain sequence of nucleotides called a PAM site, or protospacer adjacent motif. This sequence is recognised by the Cas protein and is thus necessary for it to bind to the sgRNA. The sgRNA recognises the target DNA, thereby acting as an intermediate between the Cas protein and the target. The most common PAM site is NGG where N is any nucleotide.</p>
−
Yeasts are an important expression host for the production of many recombinant proteins. The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level <sup>[1]</sup>. The promoters we have decided to use are MET25, GAL1, Cup1 and ADH1. All these promoters are found in Saccharomyces cerevisiae and therefore make excellent choices for our project which deals with prion aggregation in yeast.
+
    <p>The target DNA must be carefully chosen in order to avoid accidentally targeting genes important to the host organism, in this case <i>Saccharomyces cerevisiae</i>. CRISPR-Cas systems have been effectively employed in yeast hosts on a number of occasions, indicating that our intentions to do the same have a good chance of success (DiCarlo, 2013; 2015; Smith 2016).</p>
−
</p>
+
  </div>
−
<p>
+
−
The first promotor utilized in our project design was the MET25 promoter. The MET25 promoter is repressed when cells are grown in the presence of methionine <sup>[2]</sup>. In previous experiments, the MET25 promoter has been used for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae <sup>[3]</sup>. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA <sup>[3]</sup>. Furthermore, the MET25 catalyzes the synthesis of homocysteine from O-acetylhomoserine <sup>[4]</sup>.
+
−
</p>
+
−
<p>
+
−
Secondly, the GAL1 promoter was also used in our experiments. Expression of the GAL1 gene in S. cerevisiae is strongly repressed by growth on glucose. It is shown that two sites within the GAL1 promoter mediate glucose repression <sup>[5]</sup>. The promoter is tightly repressed by glucose and is strongly induced by galactose <sup>[6]</sup>.
+
−
</p>
+
−
<p>
+
−
The use of the Cup1 was also essential to the use of our experiments. The CUP1 promoter is a yeast metallothionein gene promoter, and may be tightly modulated by copper <sup>[7]</sup>. It is inducible by adding copper to the medium <sup>[8]</sup>.
+
−
</p>
+
−
<p>
+
−
The final crucial promoter to our experiment is alcohol dehydrogenase I (ADH1). The ADH1 promoter is a widely used constitutive yeast promoter <sup>[9]</sup>. On glucose, activity of the ADH1 promoter decreases during late exponential, ethanol production growth phase <sup>[10]</sup>. A full-length ADH1 promoter would be suitable for conditional expression of genes at high glucose concentrations <sup>[11]</sup>.
+
−
</p>
+
−
</div>
+
 
</div>
 
</div>
 +
 
<div class="wcontent-box">
 
<div class="wcontent-box">
−
<div class="wcontent-title">
+
  <div class="wcontent-title">
−
References
+
  References
−
</div>
+
  </div>
−
<div class="wcontent">
+
  <div class="wcontent">
−
<ol>
+
    <p>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. <i>Science</i> Feb 15;339(6121):819-23.</p>
−
<li>Weinhandl, K., Winkler, M., Glieder, A., and Camattari, A. (2014). Carbon source dependent promoters in yeasts. <i>Microbial Cell Factories, 13:5.</i>
+
    <p>DiCarlo, J.E. et al., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. <i>Nucleic acids research</i>, 41(7), pp.4336–43.</p>
−
</li>
+
    <p>DiCarlo, J.E. et al., 2015. Safeguarding CRISPR-Cas9 gene drives in yeast. <i>Nature Biotechnology</i>, 33(12), pp.1250–1255.</p>
−
<li> Mumberg, D., Müller, R., and Funk, M. (1994). Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. <i>Nucleic Acids Research, 22:25.</i> 5767-5768.
+
    <p>Doudna, J.A. & Charpentier, E., 2014. The new frontier of genome engineering with CRISPR-Cas9. <i>Science</i>, 346(6213), pp.1258096–1258096.</p>
−
</li>
+
    <p> Jinek, M. et al., 2012. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. <i>Science</i>, 337(6096), p.816 LP-821.</p>
−
<li>Solow, S. P., Sengbusch, J., and Laird, M. W. (2005). Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae. <i>Biotechnology Progress, 21:2.</i> 617-620.
+
    <p>Smith, J.D. et al., 2016. Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. <i>Genome Biology</i>, 17(1), p.45.</p>
−
</li>
+
  </div>
−
<li>Thomas, D., Cherest, H., and Surdin-Kerjan, Y. (1989). Elements Involved in S-Adenosylmethionine-Mediated Regulation of the Saccharomyces cerevisiae MET25 Gene. <i>Molecular and Cellular Biology, 9:8.</i> 3292-3298.
+
−
</li>
+
−
<li>Flick, J. S., and Johnston, M. (1990). Two Systems of Glucose Repression of the GAL] Promoter in Saccharomyces cerevisiae. <i>Molecular and Cellular Biology, 10:9.</i> 4747-4769.
+
−
</li>
+
−
<li>Mumberg, D., Müller, R., and Funk, M. (1994). Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. <i>Oxford University Press 22:25.</i> 5767-5768.
+
−
</li>
+
−
<li>Mascorro-Gallardo, J.O., Covarrubias, A. A., and Gaxiola, R. (1996). Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae. <i>Gene, 172:1.</i> 169-170.  
+
−
</li>
+
−
<li>Koller, A., Valesco, J., and Subramani, S. (2000). The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris. <i>Yeast, 16.</i> 651-656.
+
−
</li>
+
−
<li>Weinhandl, K, Winkler, M., Glieder, A., Camattari, A. (2014). Carbon source dependent promoters in yeasts. <i>Microbial Cell Factories, 13:5.</i>  
+
−
</li>
+
−
<li>Ruohonen, L., Aalto, M. K., Keänen, S. (1995). Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. <i>Journal of biotechnology, 39:3.</i> 193-20.
+
−
</li>
+
−
<li>Partow, S., Siewers, V., Bjøm, S., Nielsen, J., Jérôme, M. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. <i>Yeast, 27.</i> 955-964.
+
−
</li>
+
−
</ol>
+
−
+
 
</div>
 
</div>
 +
 
</body>
 
</body>
 
</html>
 
</html>
  
 
{{Template:Waterloo/Footer}}
 
{{Template:Waterloo/Footer}}

Latest revision as of 02:52, 20 October 2016

Lab and Design
Math Modelling

CRISPRi

Summary

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.

Application for PriON to PriOFF

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.

dCas9

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.

sgRNAs

All sgRNAs must start with a certain sequence of nucleotides called a PAM site, or protospacer adjacent motif. This sequence is recognised by the Cas protein and is thus necessary for it to bind to the sgRNA. The sgRNA recognises the target DNA, thereby acting as an intermediate between the Cas protein and the target. The most common PAM site is NGG where N is any nucleotide.

The target DNA must be carefully chosen in order to avoid accidentally targeting genes important to the host organism, in this case Saccharomyces cerevisiae. CRISPR-Cas systems have been effectively employed in yeast hosts on a number of occasions, indicating that our intentions to do the same have a good chance of success (DiCarlo, 2013; 2015; Smith 2016).

References

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.

DiCarlo, J.E. et al., 2013. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research, 41(7), pp.4336–43.

DiCarlo, J.E. et al., 2015. Safeguarding CRISPR-Cas9 gene drives in yeast. Nature Biotechnology, 33(12), pp.1250–1255.

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.

Smith, J.D. et al., 2016. Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design. Genome Biology, 17(1), p.45.