2018 Fiscal Year Annual Research Report
Development of in vitro transcription networks using CRISPR/Cas9 and microfluidic technologies
| Project/Area Number |
17F17796
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| Research Institution | The University of Tokyo |
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Principal Investigator |
藤井 輝夫 東京大学, 生産技術研究所, 教授 (30251474)
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| 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
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| Project Period (FY) |
2017-11-10 – 2020-03-31
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| Keywords | CRISPR/Cas9 / Reaction Network / Fluorescence / Microfluidics |
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| Outline of Annual Research Achievements |
1. Application of One-pot assay to a mutant Cas9
In order to see if our fluorescent assay can quantitatively characterize a mutant Cas9. It is a variant of Cas9 that was engineered to have an expanded range of PAM motif. We designed 3 additional fluorescent beacons to bearing those PAM, and tested the kinetics of the wild type Cas9 and the mutant Cas9 (Cas9NG). After adjusting our assay to match their experimental conditions (temperature, large excess of guide of Cas9, pre-incubation of guide and Cas9), we find a very good match between time courses of cleavage collected by gel and by fluorescence. This comforts our assay as a powerful method to quickly and quantitatively quantify kinetics of Cas9
2. Control of sgRNA activity with secondary structures
We have continued working on understanding how secondary structures of the guides affect their loading kinetics by Cas9. After testing 100+ guides (which was made possible by the fluorescent assay described above), we found a weak spot in the guide RNA whose secondary structure is essential for the guide to be loaded by Cas9. We then rebuilt the guide around this weak spot, installing competing secondary structures to control its affinity for Cas9 with an input RNA. As a proof of principle, we made guides responding to miRNAs that are highly enriched in cardiovascular cells, pancreatic cancer cells, scar tissue, psoriasis skin cells or white blood cells. We benchmarked those guides with our one-pot assay, and find that they display excellent activation, low cross-talk and limited leakage.
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| Current Status of Research Progress |
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 now testing a large number of guide against the mutant Cas9 to verify the robustness of the assay. We are also investigating in more details, and carrying out more controls, on the regulation of sgRNA with exogenous miRNA. We do not see particular problems so far.
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| Strategy for Future Research Activity |
In view of the excellent results obtained during FY2018, we are now using the remaining 6 months to wrap up the work and publish it in journal papers. We plan 3 papers: a paper describing a variant of the fluorescent assay to reduce the cost of fluorophore, one papers demonstrating the fluorescent assay for characterizing a mutant Cas9, and a paper showing how miRNA can modulate the activity of the sgRNA.
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Research Products
(1 results)
