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 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).

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.

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

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.

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 our gRNA design page for detailed information. Please use this page to write about anything related to safety in your project.

Our Lab

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!