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

Research Project

Project/Area Number 16K18821
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeMulti-year Fund
Research Field Integrative animal science
Research InstitutionTokai 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
Project Status Completed (Fiscal Year 2017)
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)
KeywordsCRISPR/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).

Report

(3 results)
  • 2017 Annual Research Report   Final Research Report ( PDF )
  • 2016 Research-status Report
  • Research Products

    (14 results)

All 2018 2017 2016 Other

All Int'l Joint Research (1 results) Journal Article (3 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 3 results,  Open Access: 3 results) Presentation (8 results) (of which Int'l Joint Research: 3 results,  Invited: 1 results) Book (1 results) Patent(Industrial Property Rights) (1 results) (of which Overseas: 1 results)

  • [Int'l Joint Research] University of Nebraska, Medical Center(米国)

    • Related Report
      2017 Annual Research Report
  • [Journal Article] Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors2018

    • Author(s)
      Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M
    • Journal Title

      Nat Protoc.

      Volume: 13 Issue: 1 Pages: 195-215

    • DOI

      10.1038/nprot.2017.153

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases2018

    • Author(s)
      Ohtsuka Masato、Sato Masahiro、Miura Hiromi、Takabayashi Shuji、Matsuyama Makoto、Koyano Takayuki、Arifin Naomi、Nakamura Shingo、Wada Kenta、Gurumurthy Channabasavaiah B.
    • Journal Title

      Genome Biology

      Volume: 19 Issue: 1 Pages: 1-15

    • DOI

      10.1186/s13059-018-1400-x

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [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
    • Journal Title

      Genome Biology

      Volume: 18 Issue: 1 Pages: 92-92

    • DOI

      10.1186/s13059-017-1220-4

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Rescue of the albino phenotype in mice using GONAD method2017

    • Author(s)
      Hiromi Miura, Naomi Arifin, Masahiro Sato, Masato Ohtsuka
    • Organizer
      International Pigment Cell Conference 2017(IPCC2017)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Development of mouse model suitable for in vivo genome editing efficiency studies2017

    • Author(s)
      Hiromi Miura, Yutaka Inagaki, Channabasavaiah B Gurumurthy, Masahiro Sato, Masato Ohtsuka
    • Organizer
      14th Transgenic Thecnology Meeting
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] CRISPR solution for creating Cre-LoxP genetics resources2017

    • Author(s)
      Rolen M Quadros, Hiromi Miura, Masato Ohtsuka, Donald W Harms, Channabasavaiah B Gurumurthy
    • Organizer
      Genome Engineering: The CRISPR-Cas Revolution(CSHL Meetings & Courses Program)
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] In vivoゲノム編集効率評価系モデルマウスの開発2017

    • Author(s)
      三浦 浩美、稲垣 豊、佐藤 正宏、大塚 正人
    • Organizer
      第40回日本分子生物学会年会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 新規遺伝子改変マウス作製方法:PITT, Easi-CRISPR, GONAD2017

    • Author(s)
      大塚正人
    • Organizer
      第3回川島カンファレンス
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] Easi-CRISPR:高効率なノックインマウス作製法2016

    • Author(s)
      三浦 浩美、Gurumurthy Channabasavaiah、相田 知海、稲垣 豊、佐藤 正宏、佐藤 健人、大塚 正人
    • Organizer
      第39回日本分子生物学会年会
    • Place of Presentation
      パシフィコ横浜(神奈川県横浜市)
    • Year and Date
      2016-11-30
    • Related Report
      2016 Research-status Report
  • [Presentation] 長い一本鎖DNAを用いたノックインマウス作製2016

    • Author(s)
      三浦 浩美、Channabasavaiah Gurumurthy、稲垣 豊、大塚 正人
    • Organizer
      平成28年度新学術領域研究「学術研究支援基盤形成【先端モデル動物支援プラットフォーム】」若手支援技術講習会
    • Place of Presentation
      蓼科グランドホテル滝の湯(長野県茅野市)
    • Year and Date
      2016-09-14
    • Related Report
      2016 Research-status Report
  • [Presentation] Easi-CRISPR:長鎖一本鎖DNAを用いた高効率ノックイン法2016

    • Author(s)
      大塚正人,相田知海,三浦浩美,Channabasavaiah Gurumurthy
    • Organizer
      日本ゲノム編集学会第1回大会
    • Place of Presentation
      広島国際会議場(広島県広島市)
    • Year and Date
      2016-09-06
    • Related Report
      2016 Research-status Report
  • [Book] PITT法によるターゲットトランスジェニックマウス作製 (動物/疾患モデルの作製技術・病態解析・評価手法)2017

    • Author(s)
      大塚正人、三浦浩美
    • Total Pages
      428
    • Publisher
      技術情報協会
    • Related Report
      2017 Annual Research Report
  • [Patent(Industrial Property Rights)] Dna editing using single-stranded dna2016

    • Inventor(s)
      CB GURU, H Miura, M Ohtsuka
    • Industrial Property Rights Holder
      Nebraska Univ., Tokai Univ.
    • Industrial Property Rights Type
      特許
    • Filing Date
      2016-06-03
    • Related Report
      2016 Research-status Report
    • Overseas

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Published: 2016-04-21   Modified: 2022-06-07  

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