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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Project Status |
Completed (Fiscal Year 2017)
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Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2017: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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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 / CRISPR / ssDNA / ノックイン / コンディショナルノックアウト |
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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Report
(3 results)
Research Products
(14 results)
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[Journal Article] Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins2017
Author(s)
Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB
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Journal Title
Genome Biology
Volume: 18
Issue: 1
Pages: 92-92
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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