研究課題/領域番号 |
17F17796
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研究機関 | 東京大学 |
研究代表者 |
藤井 輝夫 東京大学, 生産技術研究所, 教授 (30251474)
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研究分担者 |
BACCOUCHE ALEXANDRE 東京大学, 生産技術研究所, 外国人特別研究員
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研究期間 (年度) |
2017-11-10 – 2020-03-31
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キーワード | CRISPR/Cas9 / Reaction Network / fluorescence / microfluidics |
研究実績の概要 |
In this JSPS project, we aim to harness the enzyme CRISPR/Cas9 to program in vitro reaction networks with novel dynamics. In order to know the kinetics of the reaction network, it is crucial that we are able to monitor the activity of Cas9 in real time and quantitatively. To that end, during FY2017 we have optimized a fluorescence assay to measure the activity of Cas9. The assays consists of double stranded DNA that is targeted by Cas9. Upon binding and cleavage by Cas9, the top strand of the duplex is released, which is generated a fluorescence signal.
Along the way, we have devised a one-pot assay for assessing the activity of a RNA guide: it takes as input a dsDNA fragment (which can be commercially ordered) and returns a fluorescence signal showing the kinetics of the RNA guide encoded by the dsDNA fragment. We believe this discovery is noticeable and we are taking action to exploit it - through a paper and/or a patent.
Lastly, we have started moving the assay into microfluidic droplets - in view of collecting high-resolution data on the kinetics of Cas9. We have performed experiments showing that Cas9 retains its activity in droplets, and that the fluorescence assay is still functional.
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現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The research project is progressing satisfactorily. We are equipped with a fluorescent assay for Cas9, which allows us to prove quantitatively into its kinetics. We have also a one-pot assay, which considerably speeds up developpement of RNA guide. Lastly the assay appears to work in droplets. Put simply, we do not face particular problems.
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今後の研究の推進方策 |
For FY2018, we want to understand how the structure of a RNA guide affects its loading and activity in the Cas9:sgRNA. Then, we will start implementing in vitro reaction networks such as exponential kinetics, multistability, etc. One particular area of interest is to build an event-recorder with a switch, which measures how long a biochemical circuit has been active and stops the circuit when it has been active for too long.
And we will study perform competitive kinetics assays of Cas9 in droplets. Multiplexed programming of Cas9 (where several guides concurrently use Cas9) is of great interest to biotechnologists, but kinetics data has been lacking. We aim to bridge that gap by collecting high resolution data with our droplet platform.
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