Team:Stanford/Description
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Background
The CRISPR/Cas9 system is a new tool that has tremendous power for the future of bioengineering and synthetic biology. A number of projects have successfully harnessed this technology in applications spanning both gene expression manipulation and DNA detection. While historically DNA detection has been carried out via polymerase chain reaction (PCR) amplification, the specificity and adaptability of the Cas9 protein to selectively target DNA has opened the window to a new and streamlined strategy for detecting the presence of DNA fragments in vivo.
The 2015 Peking University iGEM team designed a split luciferase dual-dCas9 system that effectively targets fragments of DNA [1]. In their system, two dCas9s simultaneously bind different but neighboring sections of the target DNA, and in doing so bring together two halves of a luciferase molecule for visible detection. This project was successfully executed and optimized to reliably and adaptably detect DNA.
Our project builds on this dual dCas9 system by using a transcriptional-based reporter system. Instead of split luciferase as an indicator, in our system the proximity of dCas9 binding brings together a larger protein complex (adapted from the yeast two-hybrid system) and initiates transcription of red fluorescent protein (RFP). While this initial design accomplishes a similar goal to the 2015 Peking project, the key difference is in the adaptability of transcriptional activation as a reporter—instead of luminescence as the sole output of the system, our system can be expanded beyond detection by exchanging the RFP output for other relevant proteins for applications [hyperlink to applications] in targeted therapeutics, bacterial strain engineering, and beyond.
The System and its Scientific Basis
TBD
TBD
Three Plasmid System
TBD
Targeting GFP Plasmid
TBD
sgRNA linear fragment design
TBD
Applications
TBD