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

• 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
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥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)
• 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).

Research Products

• [Int'l Joint Research] University of Nebraska, Medical Center(米国)
• [Journal Article] Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors (2018)
  • 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
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases (2018)
  • 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
  • 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 ribonucleoproteins (2017)
  • 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
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Rescue of the albino phenotype in mice using GONAD method (2017)
  • Author(s): Hiromi Miura, Naomi Arifin, Masahiro Sato, Masato Ohtsuka
  • Organizer: International Pigment Cell Conference 2017(IPCC2017)
  • Int'l Joint Research
• [Presentation] Development of mouse model suitable for in vivo genome editing efficiency studies (2017)
  • Author(s): Hiromi Miura, Yutaka Inagaki, Channabasavaiah B Gurumurthy, Masahiro Sato, Masato Ohtsuka
  • Organizer: 14th Transgenic Thecnology Meeting
  • Int'l Joint Research
• [Presentation] CRISPR solution for creating Cre-LoxP genetics resources (2017)
  • 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)
  • 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
• [Presentation] 新規遺伝子改変マウス作製方法:PITT, Easi-CRISPR, GONAD (2017)
  • Author(s): 大塚正人
  • Organizer: 第3回川島カンファレンス
  • 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
• [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
• [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
• [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
• [Patent(Industrial Property Rights)] Dna editing using single-stranded dna (2016)
  • 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
  • Overseas