Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Mashiko, Daisuke; Fujihara, Yoshitaka; Satouh, Yuhkoh; Miyata, Haruhiko; Isotani, Ayako; Ikawa, Masahito

doi:https://doi.org/10.1038/srep03355

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Mashiko, Daisuke; Fujihara, Yoshitaka; Satouh, Yuhkoh et al.

Scientific Reports, 2013, 3, 3355

Number of Access:658（2025-08-22 01:06 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/78604

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Title	Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
Creator	CreatorName Mashiko, Daisuke
	NRID 1000070578848 1000070578848 CreatorName Fujihara, Yoshitaka
	NRID 1000040545571 1000040545571 CreatorName Satouh, Yuhkoh
	NRID 1000050604732 1000050604732 researchmap permalink hmiyata hmiyata Scopus Author ID 37161837000 37161837000 ORCID 0000-0003-4758-5803 0000-0003-4758-5803 CreatorName Miyata, Haruhiko
	NRID 1000020444523 1000020444523 CreatorName Isotani, Ayako
	NRID 1000020304066 1000020304066 ORCID 0000-0001-9859-6217 0000-0001-9859-6217 researchmap permalink read0054910 read0054910 Scopus Author ID 16689054500 16689054500 CreatorName Ikawa, Masahito
Description	Mashiko, D., Fujihara, Y., Satouh, Y. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3, 3355 (2013). https://doi.org/10.1038/srep03355
Abstract	CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.
Publisher	Nature Research
Source Title	Scientific Reports
Volume (Issue)	3
Page	3355
Date of Issued	2013-11-27
Language	English
Handle URL	https://hdl.handle.net/11094/78604
eISSN	20452322
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1038/srep03355
Relation.isIdenticalTo	PMID 24284873
Access Rights	open access
CopyRight	© 2013 The Author(s). This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCTERMS.bibliographicCitation	Scientific Reports. 2013, 3, p. 3355
DC.title	Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
DC.creator	Mashiko, Daisuke
	Fujihara, Yoshitaka
	Satouh, Yuhkoh
	Miyata, Haruhiko
	Isotani, Ayako
	Ikawa, Masahito
DC.publisher	Nature Research
DC.language' scheme='DCTERMS.RFC1766	English
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2013-11-27
DC.identifier	https://doi.org/10.1038/srep03355
DC.description	Mashiko, D., Fujihara, Y., Satouh, Y. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3, 3355 (2013). https://doi.org/10.1038/srep03355
DCTERMS.abstract	CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.
DC.rights	© 2013 The Author(s). This article is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
DC.format	78604
citation_title	Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
citation_author	Mashiko, Daisuke
	Fujihara, Yoshitaka
	Satouh, Yuhkoh
	Miyata, Haruhiko
	Isotani, Ayako
	Ikawa, Masahito
citation_publisher	Nature Research
citation_language	English
citation_date	2013-11-27
citation_journal_title	Scientific Reports
citation_volume	3
citation_firstpage	3355
citation_doi	https://doi.org/10.1038/srep03355
citation_pmid	24284873
citation_public_url	https://hdl.handle.net/11094/78604

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

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