| Project/Area Number |
26640064
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Laboratory animal science
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| Research Institution | University of Yamanashi |
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Principal Investigator |
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Project Status |
Completed (Fiscal Year 2015)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2015: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2014: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
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| Keywords | ゲノム編集技術 / CRISPR/Cas9 / ノックイン法 / ゼブラフィッシュ / 遺伝子破壊 / 遺伝子置換 / 発生工学解析技術 / モデル生物 |
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| Outline of Final Research Achievements |
CRISPR/Cas9 system provides a powerful genetic tool for genome editing in zebrafish. However, it is still difficult to manipulate precise integration of exogenous DNA at the targeted genomic locus. We developed CRISPR/Cas9-mediated integration of the EGFP reporter containing short homologous sequences flanking the target locus. We succeeded in the efficient genomic integration of EGFP gene into the target site and the genome modification was heritable, presenting a simple CRISPR/Cas9-mediated knock-in system.
We tried to develop a ready-to-used CRISPR/Cas9 that composed of synthetic crRNA and tracrRNA with recombinant Cas9 protein. We found that the injection of crRNAs, tracrRNA and Cas9 protein rapidly induced genome modifications compared with the injection of sgRNAs and Cas9 mRNA. Furthermore, we demonstrated that this ready-to-used CRISPR/Cas9 is acceptable for the visualization of endogenous target gene expression using the EGFP reporter in zebrafish.
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