Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes
•Novel CRISPR-Cas vectors with a Cas9 fused to a part of mouse Cdt1.•Nuclear localisation of Cdt1-fused Cas9 in mammalian cells and embryos.•High efficiency in megabase-scale large deletion and knock-in mouse production. Genetically modified mouse models are essential for in vivo investigation of ge...
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Published in | Methods (San Diego, Calif.) Vol. 191; pp. 23 - 31 |
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Main Authors | , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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United States
Elsevier Inc
01.07.2021
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Abstract | •Novel CRISPR-Cas vectors with a Cas9 fused to a part of mouse Cdt1.•Nuclear localisation of Cdt1-fused Cas9 in mammalian cells and embryos.•High efficiency in megabase-scale large deletion and knock-in mouse production.
Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations. |
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AbstractList | Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations. •Novel CRISPR-Cas vectors with a Cas9 fused to a part of mouse Cdt1.•Nuclear localisation of Cdt1-fused Cas9 in mammalian cells and embryos.•High efficiency in megabase-scale large deletion and knock-in mouse production. Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations. |
Author | Matoba, Shogo Suzuki, Hayate Murata, Kazuya Mizuno-Iijima, Saori Iki, Natsumi Obata, Yuichi Ogura, Atsuo Kato, Kanako Nakashiba, Toshiaki Dinh, Tra Thi Huong Mizuno, Seiya Yagami, Ken-ichi Takahashi, Satoru Ishida, Miyuki Daitoku, Yoko Ayabe, Shinya Ikeda, Yoshihisa Yoshiki, Atsushi Tanimoto, Yoko Sugiyama, Fumihiro Nakashima, Kenichi Ibrahim, Elzeftawy Abdelaziz Elsayed Hasegawa, Yoshikazu Okada-Iwabu, Miki Le, Hoai Thu Miwa, Yoshihiro Hamada, Yuko Hamada, Michito Iwabu, Masato |
Author_xml | – sequence: 1 givenname: Saori surname: Mizuno-Iijima fullname: Mizuno-Iijima, Saori organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 2 givenname: Shinya surname: Ayabe fullname: Ayabe, Shinya email: shinya.ayabe@riken.jp organization: Experimental Animal Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 3 givenname: Kanako surname: Kato fullname: Kato, Kanako organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 4 givenname: Shogo surname: Matoba fullname: Matoba, Shogo organization: Bioresource Engineering Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 5 givenname: Yoshihisa surname: Ikeda fullname: Ikeda, Yoshihisa organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 6 givenname: Tra Thi Huong surname: Dinh fullname: Dinh, Tra Thi Huong organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 7 givenname: Hoai Thu surname: Le fullname: Le, Hoai Thu organization: Ph.D Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 8 givenname: Hayate surname: Suzuki fullname: Suzuki, Hayate organization: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 9 givenname: Kenichi surname: Nakashima fullname: Nakashima, Kenichi organization: Gene Engineering Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 10 givenname: Yoshikazu surname: Hasegawa fullname: Hasegawa, Yoshikazu organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 11 givenname: Yuko surname: Hamada fullname: Hamada, Yuko organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 12 givenname: Yoko surname: Tanimoto fullname: Tanimoto, Yoko organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 13 givenname: Yoko surname: Daitoku fullname: Daitoku, Yoko organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 14 givenname: Natsumi surname: Iki fullname: Iki, Natsumi organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 15 givenname: Miyuki surname: Ishida fullname: Ishida, Miyuki organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 16 givenname: Elzeftawy Abdelaziz Elsayed surname: Ibrahim fullname: Ibrahim, Elzeftawy Abdelaziz Elsayed organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 17 givenname: Toshiaki surname: Nakashiba fullname: Nakashiba, Toshiaki organization: Experimental Animal Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 18 givenname: Michito surname: Hamada fullname: Hamada, Michito organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 19 givenname: Kazuya surname: Murata fullname: Murata, Kazuya organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 20 givenname: Yoshihiro surname: Miwa fullname: Miwa, Yoshihiro organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 21 givenname: Miki surname: Okada-Iwabu fullname: Okada-Iwabu, Miki organization: Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 22 givenname: Masato surname: Iwabu fullname: Iwabu, Masato organization: Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan – sequence: 23 givenname: Ken-ichi surname: Yagami fullname: Yagami, Ken-ichi organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 24 givenname: Atsuo surname: Ogura fullname: Ogura, Atsuo organization: Bioresource Engineering Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 25 givenname: Yuichi surname: Obata fullname: Obata, Yuichi organization: Gene Engineering Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 26 givenname: Satoru surname: Takahashi fullname: Takahashi, Satoru organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 27 givenname: Seiya surname: Mizuno fullname: Mizuno, Seiya email: konezumi@md.tsukuba.ac.jp organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan – sequence: 28 givenname: Atsushi surname: Yoshiki fullname: Yoshiki, Atsushi organization: Experimental Animal Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan – sequence: 29 givenname: Fumihiro surname: Sugiyama fullname: Sugiyama, Fumihiro organization: Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/32334080$$D View this record in MEDLINE/PubMed |
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Keywords | CRISPR Genome editing Cas9 Knock-in Genetically engineered mouse model |
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Snippet | •Novel CRISPR-Cas vectors with a Cas9 fused to a part of mouse Cdt1.•Nuclear localisation of Cdt1-fused Cas9 in mammalian cells and embryos.•High efficiency in... Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced... |
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SubjectTerms | Animals Cas9 Cell Cycle Proteins CRISPR CRISPR-Cas Systems - genetics DNA-Binding Proteins Gene Editing Gene Knock-In Techniques Genetically engineered mouse model Genome editing HEK293 Cells Humans Knock-in Mice Zygote |
Title | Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes |
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