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Team:KAIST Korea/Project
From 2013.igem.org
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Overall project
The widely used gene knock-out strategies are based upon a mere ‘on-off’ control and more complex regulation is impossible. On the other hand, knock-down strategies are more versatile in usage but the current techniques are vastly laborious for systematic control and pose difficulty in multiple targeting because of the intricate design and scar accumulation.
iGEM KAIST 2013 seeks to take the yet-to-be-perfected genetic regulation technology to a new level: The RAPTOR (RNA Associated Prokaryotic Transcript Optimization with CRISPR) technology. RAPTOR uses CRISPR type III system for gene regulation. CRISPR type III targets and cleaves specific mRNA sequences through complementary binding of crRNA and nuclease activity of Cmr protein complex. RAPTOR exploits this nature of CRISPR for systematically down regulating the mRNA level of specific genes.
Due to the characteristics of RAPTOR, one of the most useful applications of this technology can be biosynthesis pathway regulation. Programmed crRNA expression allows fine-tuning of target mRNA expression levels and can precisely regulate enzyme concentrations. Not only fine-tuning but multiplex engineering is also possible through simultaneous expression of different crRNAs. Thus, multiple enzymes can be targeted in pathway control.
As a proof of concept we sought to regulate the lycopene pathway that is incorporated into the genome of Escherichia coli. The efflux of lycopene precursors is optimally reduced through lowering the mRNA levels of enzymes that synthesize non-lycopene end products. Through observation of increased lycopene yield in the E.coli, potentiality of RAPTOR technology can be validated.
