Team:SUSTC-Shenzhen/Safety

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=Safety Concerns About the Project
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=Safety Concerns About the Project=
==Control the expression of Cas9==
==Control the expression of Cas9==
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Though CRISPR/Cas genome editing techniques have high specificity due to the gRNA, it still has off-target effect which may lead to host genome mutagenesis and chromosomal disorders, cytotoxicity, genotoxicity, or oncogenesis, especially when Cas9 is chronically expressed. Thus, we want to improve our genetic circuit so that we can switch on and off the permanent transfected Cas9 at any time. Here, we use the TetON operon, or precisely Tet-On 3G System. The Tet-On 3G System is the third generation of tetracyclin inducible gene expression systems developed for mammalian cells (Clontech). Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G) will highly express the GOI, when cultured in the presence of doxycycline (Dox), a synthetic tetracycline derivative. Figure 1 demonstrates the mechanism of Tet-ON 3G system (Clontech).
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Though CRISPR/Cas genome editing techniques have high specificity due to the gRNA, it still has off-target effect. Chronic expression of Cas9 may amplify such effect and lead to host genome mutagenesis and chromosomal disorders, cytotoxicity, genotoxicity, or oncogenesis. Thus, we want to improve our genetic circuit to reduce the possible chronic side effect while retain its function. Our solution is to utilize chemical inducible system to switch on Cas9 gene when needed and switch it off when not needed. Here, we put the stably transfected Cas9 under the control of Tet-On 3G system, which is the third generation of tetracyclin inducible gene expression systems developed for mammalian cells (Clontech). Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G) will highly express the GOI, when cultured in the presence of doxycycline (Dox), a synthetic tetracycline derivative. Figure 1 illustrates the mechanism of Tet-ON 3G system (Clontech).
<center>{{SUSTC-Image|wiki/images/8/82/SUSTC-Shenzhen-Project-Tet-On.png}}</center>
<center>{{SUSTC-Image|wiki/images/8/82/SUSTC-Shenzhen-Project-Tet-On.png}}</center>
<center>Figure 1 Mechanism of Tet-ON 3G operon</center>
<center>Figure 1 Mechanism of Tet-ON 3G operon</center>
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The TRE3G promoter has very low background expression level. This highly reduces potential risks of accidental injury due to the off-target effect of CRISPR/Cas system (Figure 12). Also, it is very sensitive to the concentration of doxycycline and functions like an exponential function over the Dox concentration. And the Dox concentration required for the induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies which also reduces the usage of antibiotics. So it is ideal to use Tet-ON 3G as a mammalian gene expression controller.  
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The TRE3G promoter has very low background expression level when Dox is absent. It is the lowest we have found for mammalian inducible system. This greatly reduces potential risks of off-target effect of CRISPR/Cas system (Figure 2). Furthermore, the Dox concentration required for the induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies which also reduces the usage of antibiotics (Figure 3). Such on-demand expression system with minimum leaking will greatly alleviate the potential safety risk of the CRISPR/Cas and enable the optimizing of the Cas9 expression level and timing in the potential clinical application. So it is ideal to use Tet-ON 3G as a mammalian gene expression controller for this project.  
   
   
<center>{{SUSTC-Image|wiki/images/b/b6/SUSTC-Shenzhen-Project-TetON-low_background.jpg}}</center>
<center>{{SUSTC-Image|wiki/images/b/b6/SUSTC-Shenzhen-Project-TetON-low_background.jpg}}</center>
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==Control the gRNA expression by A-B toxin-based shuttle==
==Control the gRNA expression by A-B toxin-based shuttle==
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To further confirm the safety of our system, we want to make one more step: controlling the expression of gRNA. Unlike many other recent researches which introduce gRNA together with Cas9, we introduce gRNA by using A-B toxin-based shuttle. Even Cas9 expression is accidentally turned on, Cas9 won’t work without gRNA. Also, the plasmid encoding gRNA is only transiently transfected into the cell, which can’t replicate during cell replication and will lost 2-3 days after transfection. This will further reduce the off-target effect of the system.
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To further improve the safety of our system, we want to control the timing and location of the expression of gRNA. Unlike many other recent researches which introduce gRNA together with Cas9, we introduce gRNA by using A-B toxin-based shuttle. Even Cas9 expression is accidentally turned on, Cas9 won’t work without gRNA. As the plasmid encoding gRNA is only transiently transfected into the cell, which can’t replicate during cell replication and will lost 2-3 days after transfection in typical cell culture. In addition, it will be readily targeted to specific cell type by conjugating either ligand or antibody specifically for the cell type. This will further reduce the off-target effect of the system.
==Carefully designed gRNA to decrease off-target effect==
==Carefully designed gRNA to decrease off-target effect==
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The gRNA we designed has been matched to human genome and has few off-target sites. See our gRNA design page for detailed information.
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The gRNA we designed has been matched to human genome and has few off-target sites.  
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Please use this page to write about anything related to safety in your project.
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=Our Lab=
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<a href="/Team:SUSTC-Shenzhen/gRNA_Design" class="btn btn-success btn-lg" style="color:#fff">See how we optimized our gRNA design to reduce off-target effect</a>
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</html>
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Use this section to tell us about your laboratory. Where is it located? What sort of equipment do you use every day? Have you decorated it for the summer? How do you look wearing a lab coat? Take pictures! Show off your space!
 
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Latest revision as of 03:29, 18 October 2014

Team SUSTC-Shenzhen

Safety

Doing research with responsibility

Safety Concerns About the Project

Control the expression of Cas9

Though CRISPR/Cas genome editing techniques have high specificity due to the gRNA, it still has off-target effect. Chronic expression of Cas9 may amplify such effect and lead to host genome mutagenesis and chromosomal disorders, cytotoxicity, genotoxicity, or oncogenesis. Thus, we want to improve our genetic circuit to reduce the possible chronic side effect while retain its function. Our solution is to utilize chemical inducible system to switch on Cas9 gene when needed and switch it off when not needed. Here, we put the stably transfected Cas9 under the control of Tet-On 3G system, which is the third generation of tetracyclin inducible gene expression systems developed for mammalian cells (Clontech). Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G) will highly express the GOI, when cultured in the presence of doxycycline (Dox), a synthetic tetracycline derivative. Figure 1 illustrates the mechanism of Tet-ON 3G system (Clontech).

{{{2}}}
Figure 1 Mechanism of Tet-ON 3G operon

The TRE3G promoter has very low background expression level when Dox is absent. It is the lowest we have found for mammalian inducible system. This greatly reduces potential risks of off-target effect of CRISPR/Cas system (Figure 2). Furthermore, the Dox concentration required for the induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies which also reduces the usage of antibiotics (Figure 3). Such on-demand expression system with minimum leaking will greatly alleviate the potential safety risk of the CRISPR/Cas and enable the optimizing of the Cas9 expression level and timing in the potential clinical application. So it is ideal to use Tet-ON 3G as a mammalian gene expression controller for this project.

{{{2}}}
Figure 2. Low background expression of Tet-On 3G system
{{{2}}}
Figure 3 High sensitivity of Tet-On 3G system

Control the gRNA expression by A-B toxin-based shuttle

To further improve the safety of our system, we want to control the timing and location of the expression of gRNA. Unlike many other recent researches which introduce gRNA together with Cas9, we introduce gRNA by using A-B toxin-based shuttle. Even Cas9 expression is accidentally turned on, Cas9 won’t work without gRNA. As the plasmid encoding gRNA is only transiently transfected into the cell, which can’t replicate during cell replication and will lost 2-3 days after transfection in typical cell culture. In addition, it will be readily targeted to specific cell type by conjugating either ligand or antibody specifically for the cell type. This will further reduce the off-target effect of the system.

Carefully designed gRNA to decrease off-target effect

The gRNA we designed has been matched to human genome and has few off-target sites.

See how we optimized our gRNA design to reduce off-target effect


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