Difference between revisions of "Team:Toronto/Description"

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<div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main"><h1>Wetlab</h1>
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<p>The wet lab study a LacILOV/cI repressor circuit for the purpose of controlling CRISPR gene editing. The LacILOV repressor is a light-activated fusion protein which represses downstream transcription of sgRNA necessary for CRISPR-Cas9 targeting and the cI repressor in the dark, but allows transcription in blue light. The cI repressor prevents transcription of the anti-CRISPR protein AcrIIA4spy, thus forming a complementary &quot;OFF&quot; command for the LacILOV-controlled sgRNA &quot;ON&quot; switch. The switch will be validated in E. coli cells in a circuit, using the response YFP and mCherry to measure its kinetics via fluorescence and qPCR assays. Finally, CRISPR activity will be tested by targeting the araC gene with CRISPRi for silencing, and measuring the resulting population levels in an arabinose environment. Hello World!! how is it going? Hi again!</p>
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        <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>
  
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<p>Our dry lab team is responsible for developing a model of the gene circuit designed by the wet lab. Previous studies have illustrated the stochastic nature of gene regulatory networks, indicating the complexity of interactions. Thus, stochastic model algorithms will be used to simplify the complexity of gene regulation. Specifically, the dry lab team will recreate the gene pathway and determine the rate kinetics of the many outputs of our system, using the data provided by the wet lab. Next, the dynamics of our CRISPR-Cas 9 system will be analyzed with MATLAB Symbio Library. Finally, our wet lab team will implement the newly designed genetic circuit using the optimized model created by the dry lab.</p>
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<h1>Policy &amp; Practices</h1>
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              <p>asfads</p>
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<p>While the light activated CRISPR-Cas 9 system developed in this project should provide scientists with greater control and accuracy when gene editing, its effects on a larger scale (at the level of multicellular organisms such as humans or plants) are more contested. The indefinite nature of this technology has resulted in barriers to integrating it into mainstream healthcare and policy discourse.</p>
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<h2>Our Switch</h2>
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<p>Although mathematical modeling will attempt to increase the accuracy of our gene editing system it is important to understand and address societal apprehensions to integrating it into mainstream discourse. To investigate these barriers our team will create a dialogue with individuals who represent diverse cultural, economic, and religious backgrounds. These will include local politicians, healthcare professionals, members of religious communities, and end users who will be affected by the inclusion of this technology into mainstream healthcare. Through this dialogue we hope 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. Our team also seeks to engage the broader public in dialogue on synthetic biology by making it more accessible in three ways. The first is by educating and engaging high school students through a summer camp which incorporates activities such as protein modeling, coding, and case studies. Through this camp we seek to provide practical skills to students and foster an interest in synthetic biology and introduce the social impacts of it. The second will pair scientists and artists for a daylong Iconathon event. Not only will the icons created through this event be used to enrich the currently meager synthetic biology icon repository, it will also act as a way to educate artists about synthetic biology in an interdisciplinary fashion. Finally, a five episode podcast related to synthetic biology will engage industry professionals, specialists, and our own team members to educate a wider audience.</p>
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<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>
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        <h2>Experimental Data and Computational Model</h2>
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<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>
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        <h2>Policy and Practices</h2>
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  <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.
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<li id="ref1">Canadian Biosafety Standards and Guidelines - <a href="https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html">https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html</a></li>
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<li id="ref2">Biosafety Team in the Office of Environmental Health and Safety - <a href="https://ehs.utoronto.ca/our-services/biosafety/">https://ehs.utoronto.ca/our-services/biosafety/</a></li>
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<li id="ref3">NIH Guidelines - <a href="https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html">https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html</a></li>
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<li id="ref4">iGEM Risk Groups - <a href="https://2017.igem.org/Safety/Risk_Groups">https://2017.igem.org/Safety/Risk_Groups</a></li>
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<h3>Contents</h3>
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<h3>Related Pages</h3>
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Revision as of 22:51, 1 November 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.

data

asfads

sadsadassa

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.

References

  1. Canadian Biosafety Standards and Guidelines - https://www.canada.ca/en/public-health/services/canadian-biosafety-standards-guidelines.html
  2. Biosafety Team in the Office of Environmental Health and Safety - https://ehs.utoronto.ca/our-services/biosafety/
  3. NIH Guidelines - https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment.html
  4. iGEM Risk Groups - https://2017.igem.org/Safety/Risk_Groups

General Pages

Wet Lab

Dry Lab

Human Practices

iGEM Toronto