2017 Fiscal Year Final Research Report
Easi-CRISPR for creating genetically modified mice using long ssDNA.
Project/Area Number |
16K18821
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Research Category |
Grant-in-Aid for Young Scientists (B)
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Allocation Type | Multi-year Fund |
Research Field |
Integrative animal science
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Research Institution | Tokai University |
Principal Investigator |
MIURA Hiromi 東海大学, 医学部, 特定研究員 (90599523)
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Project Period (FY) |
2016-04-01 – 2018-03-31
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Keywords | CRISPR/Cas9 / 一本鎖DNA / レポーターノックインマウス / コンディショナルノックアウトマウス |
Outline of Final Research Achievements |
CRISPR/Cas9-based genome editing technology allows to disrupt mouse genes with high efficiency, but it was still challenging to create knock-in and conditional knock out models (typically 0-10%). We previously demonstrated that long single-stranded DNA (ssDNA) (up to 500 bases) serve as very efficient donors for knock-in. In this study, we tried to generate much longer ssDNA by using original ivTRT method, and applied it for generation of reporter knock-in and conditional knock-out mouse models. As a result, up to 2000 bases of ssDNA was synthesized with the ivTRT method. By using these ssDNAs, several reporter knock-in and conditional knock-out mice were generated, which insertion efficiency was quite high (typically 30%-60%, and up to 100% in some cases). We call this method Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR).
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Free Research Field |
遺伝子工学
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