Difference between revisions of "Team:Toronto/Description"

 
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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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<h3 class="text-yellow">Light-activated gene expression</h3>
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<p>Light is an attractive mode of gene regulation that provides high spatio-temporal resolution with relatively low levels of toxicity. In order to add to the genetic toolbox, we characterized a novel light-activated gene regulation system that combines the DNA-binding region of LacI with the light inducible LOV (Light Oxygen Voltage) domain. The characterization assay was performed by measuring the fluorescence output of a LacILOV-regulated reporter under blue light illumination. We then computationally modelled the structure of our protein and identified key mutations to optimize its activity.</p>
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<h2>Our Switch</h2>
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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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<p>In order to inform future applications of our tool, we identified key stakeholders that would be impacted by potential uses of LacILOV including experts, businesses, the public and future scientists. To this end, we developed resources to promote interest and meaningful interdisciplinary dialogue between researchers and the public. This was achieved through a podcast series exploring the interaction of synthetic biology with different disciplines, a synthetic biology workshop for burgeoning scientists and an iconathon to promote collaborations between scientists and artists.</p>
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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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<p>However, in order to demonstrate the utility of our tool, we decided to focus on human gene editing, an area that would benefit from stringent gene regulation. We designed and modelled a light activated switch to control CRISPR-Cas9 activity by putting guide RNAs and anti-CRISPR proteins under LacILOV-regulated promoters. We then investigated the ethical and technical concerns of our stakeholders through an interview series involving scientists, engineers, physicians, advocacy groups and religious leaders. Based on technical feedback, we identified an accurate light delivery system as a key technical barrier to validating our tool in mammalian cultures, the next step in its translation to the clinic. We therefore designed and prototyped hardware to pave the way for our system to be used in stem cell cultures. Secondly, based on the different opinions on the ethical applications of human gene editing, we identified the need for clear ethical guidelines. Using the recommendations of the 2017 report by the National Academy of Science, we developed an ethical guide for future iGEM teams.
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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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Latest revision as of 20:44, 14 December 2017

Description

Light-activated gene expression

Light is an attractive mode of gene regulation that provides high spatio-temporal resolution with relatively low levels of toxicity. In order to add to the genetic toolbox, we characterized a novel light-activated gene regulation system that combines the DNA-binding region of LacI with the light inducible LOV (Light Oxygen Voltage) domain. The characterization assay was performed by measuring the fluorescence output of a LacILOV-regulated reporter under blue light illumination. We then computationally modelled the structure of our protein and identified key mutations to optimize its activity.

Identifying and informing stakeholders

In order to inform future applications of our tool, we identified key stakeholders that would be impacted by potential uses of LacILOV including experts, businesses, the public and future scientists. To this end, we developed resources to promote interest and meaningful interdisciplinary dialogue between researchers and the public. This was achieved through a podcast series exploring the interaction of synthetic biology with different disciplines, a synthetic biology workshop for burgeoning scientists and an iconathon to promote collaborations between scientists and artists.

Applying LacILOV to human gene editing

However, in order to demonstrate the utility of our tool, we decided to focus on human gene editing, an area that would benefit from stringent gene regulation. We designed and modelled a light activated switch to control CRISPR-Cas9 activity by putting guide RNAs and anti-CRISPR proteins under LacILOV-regulated promoters. We then investigated the ethical and technical concerns of our stakeholders through an interview series involving scientists, engineers, physicians, advocacy groups and religious leaders. Based on technical feedback, we identified an accurate light delivery system as a key technical barrier to validating our tool in mammalian cultures, the next step in its translation to the clinic. We therefore designed and prototyped hardware to pave the way for our system to be used in stem cell cultures. Secondly, based on the different opinions on the ethical applications of human gene editing, we identified the need for clear ethical guidelines. Using the recommendations of the 2017 report by the National Academy of Science, we developed an ethical guide for future iGEM teams.

General Pages

Wet Lab

Dry Lab

Human Practices

iGEM Toronto