Easi-CRISPR for creating genetically modified mice using long ssDNA.

2017 Fiscal Year Final Research Report

• Project/Area Number: 16K18821
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Integrative animal science
• Research Institution: Tokai University
• Principal Investigator: MIURA Hiromi 東海大学, 医学部, 特定研究員 (90599523)
• Project Period (FY): 2016-04-01 – 2018-03-31
• 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).

Free Research Field

遺伝子工学