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Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
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 sgRN...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842082/ https://www.ncbi.nlm.nih.gov/pubmed/24284873 http://dx.doi.org/10.1038/srep03355 |
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author | Mashiko, Daisuke Fujihara, Yoshitaka Satouh, Yuhkoh Miyata, Haruhiko Isotani, Ayako Ikawa, Masahito |
author_facet | Mashiko, Daisuke Fujihara, Yoshitaka Satouh, Yuhkoh Miyata, Haruhiko Isotani, Ayako Ikawa, Masahito |
author_sort | Mashiko, Daisuke |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3842082 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-38420822013-11-27 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 Sci Rep Article 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. Nature Publishing Group 2013-11-27 /pmc/articles/PMC3842082/ /pubmed/24284873 http://dx.doi.org/10.1038/srep03355 Text en Copyright © 2013, Macmillan Publishers Limited. All rights reserved http://creativecommons.org/licenses/by-nc-sa/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ |
spellingShingle | Article Mashiko, Daisuke Fujihara, Yoshitaka Satouh, Yuhkoh Miyata, Haruhiko Isotani, Ayako Ikawa, Masahito Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title | Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title_full | Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title_fullStr | Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title_full_unstemmed | Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title_short | Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA |
title_sort | generation of mutant mice by pronuclear injection of circular plasmid expressing cas9 and single guided rna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842082/ https://www.ncbi.nlm.nih.gov/pubmed/24284873 http://dx.doi.org/10.1038/srep03355 |
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