Difference between revisions of "Team:Toronto"
| Line 95: | Line 95: | ||
</div> | </div> | ||
<div class="content"> | <div class="content"> | ||
| − | <div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main">< | + | <div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main"><h3>Description</h3> |
| − | <p> | + | |
| − | <p> | + | <p>CRISPR-Cas9 is heralded as a revolution in gene-editing technology for making directed genome editing faster and more financially viable as a form of therapy than ever before. However, the fidelity of genetic integrations is still reliant upon the activity of repair mechanism in the host cell and the system is susceptible to off-target edits depending on the duration and level of Cas9 expression. That's why our 2017 iGEM Toronto summer team is working on developing a light-activated system that will allow spacio-temporal control of CRISPR-Cas9 activity.</p> |
| − | <p> | + | <h3>Our Switch</h3> |
| + | |||
| + | <p>Using sgRNAs (necessary for CRISPR-Cas9 targeting) and AcrIIA4 (anti-CRISPR protein) under the control of LacILOV (a novel light-activated modulator developed by the Mahadevan lab) and cI repressor respectively, we aim to create a toggle switch to allow stringent control of Cas9 activity. In the absence of blue light, LacILOV represses downstream transcription of both sgRNA and the cI repressor. The lack of cI protein expression allows AcrIIA4 to be transcribed which in turn inhibits Cas9 activity. As such, without blue light, CRISPR is essentially “OFF” lacking sgRNA and inhibited by anti-CRISPR. In the presence of blue light, however, LacILOV repression is abolished allowing sgRNAs transcription and removing anti-CRISPR inhibition (through cI expression) to facilitate CRISPR activity.</p> | ||
| + | <h3>Experimental Data and Computational Model</h3> | ||
| + | |||
| + | <p>The switch will be validated in E. coli where the kinetics of light-induced activation and repression will be characterized by spectrophotometric assays of reporter proteins (YFP and mCherry). Measurable interference of metabolic and reporter genes by dCas9 will be used to assay functional control of CRISPR activity by our toggle switch. Our computational team will then be using these experimental results to simulate a stochastic model of the gene circuit. Furthermore, the dynamics of the system will be analyzed using MATLAB Symbio Library to provide an optimized model for the wet lab.</p> | ||
| + | <h3>Policy and Practices</h3> | ||
| + | |||
| + | <p>While this light activated CRISPR-Cas9 system should provide scientists with greater control and accuracy in gene editing, the regulatory framework required to implement this technology as a normalized component of healthcare, is still lacking. To investigate the barriers that human gene editing may face, our team will conduct a systematic analysis of the socioeconomic, legal, ethical and political considerations of key shareholders impacted. This will be done by creating a dialogue through interviews and a podcast series with individuals including local politicians, healthcare professionals, members of religious communities, advocacy groups, and end users who will be affected by the inclusion of this technology into mainstream healthcare. We are also committed to outreach and education through our high school summer camp on regenerative medicine and synthetic biology, and a daylong iconathon event which will pair scientists and artists to enrich the currently meager synthetic biology icon repository. Our project aims to contribute to the body of research geared towards making CRISPR an accurate, reliable and ultimately safe clinical option as well as to provide a diverse understanding of the manner in which this technology will shape, and is shaped by, the political and social landscape of Canada.</p> | ||
</div></div><div id="tableofcontents" class="tableofcontents affix sidebar col-lg-4 hidden-xs hidden-sm hidden-md visible-lg-3"><ul class="nav"></div></div></div> | </div></div><div id="tableofcontents" class="tableofcontents affix sidebar col-lg-4 hidden-xs hidden-sm hidden-md visible-lg-3"><ul class="nav"></div></div></div> | ||
</div> | </div> | ||
</html> | </html> | ||
{{Toronto/footer}} | {{Toronto/footer}} | ||
Revision as of 23:20, 29 June 2017
Description
CRISPR-Cas9 is heralded as a revolution in gene-editing technology for making directed genome editing faster and more financially viable as a form of therapy than ever before. However, the fidelity of genetic integrations is still reliant upon the activity of repair mechanism in the host cell and the system is susceptible to off-target edits depending on the duration and level of Cas9 expression. That's why our 2017 iGEM Toronto summer team is working on developing a light-activated system that will allow spacio-temporal control of CRISPR-Cas9 activity.
Our Switch
Using sgRNAs (necessary for CRISPR-Cas9 targeting) and AcrIIA4 (anti-CRISPR protein) under the control of LacILOV (a novel light-activated modulator developed by the Mahadevan lab) and cI repressor respectively, we aim to create a toggle switch to allow stringent control of Cas9 activity. In the absence of blue light, LacILOV represses downstream transcription of both sgRNA and the cI repressor. The lack of cI protein expression allows AcrIIA4 to be transcribed which in turn inhibits Cas9 activity. As such, without blue light, CRISPR is essentially “OFF” lacking sgRNA and inhibited by anti-CRISPR. In the presence of blue light, however, LacILOV repression is abolished allowing sgRNAs transcription and removing anti-CRISPR inhibition (through cI expression) to facilitate CRISPR activity.
Experimental Data and Computational Model
The switch will be validated in E. coli where the kinetics of light-induced activation and repression will be characterized by spectrophotometric assays of reporter proteins (YFP and mCherry). Measurable interference of metabolic and reporter genes by dCas9 will be used to assay functional control of CRISPR activity by our toggle switch. Our computational team will then be using these experimental results to simulate a stochastic model of the gene circuit. Furthermore, the dynamics of the system will be analyzed using MATLAB Symbio Library to provide an optimized model for the wet lab.
Policy and Practices
While this light activated CRISPR-Cas9 system should provide scientists with greater control and accuracy in gene editing, the regulatory framework required to implement this technology as a normalized component of healthcare, is still lacking. To investigate the barriers that human gene editing may face, our team will conduct a systematic analysis of the socioeconomic, legal, ethical and political considerations of key shareholders impacted. This will be done by creating a dialogue through interviews and a podcast series with individuals including local politicians, healthcare professionals, members of religious communities, advocacy groups, and end users who will be affected by the inclusion of this technology into mainstream healthcare. We are also committed to outreach and education through our high school summer camp on regenerative medicine and synthetic biology, and a daylong iconathon event which will pair scientists and artists to enrich the currently meager synthetic biology icon repository. Our project aims to contribute to the body of research geared towards making CRISPR an accurate, reliable and ultimately safe clinical option as well as to provide a diverse understanding of the manner in which this technology will shape, and is shaped by, the political and social landscape of Canada.
