Development of in vitro transcription networks using CRISPR/Cas9 and microfluidic technologies

2017 Fiscal Year Annual Research Report

• Project/Area Number: 17F17796
• Research Institution: The University of Tokyo
• Principal Investigator: 藤井 輝夫 東京大学, 生産技術研究所, 教授 (30251474)
• Co-Investigator(Kenkyū-buntansha): BACCOUCHE ALEXANDRE 東京大学, 生産技術研究所, 外国人特別研究員
• Project Period (FY): 2017-11-10 – 2020-03-31
• Keywords: CRISPR/Cas9 / Reaction Network / fluorescence / microfluidics

Outline of Annual Research Achievements

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.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

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.

Strategy for Future Research Activity

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.