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

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...

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Main Authors: Mashiko, Daisuke, Fujihara, Yoshitaka, Satouh, Yuhkoh, Miyata, Haruhiko, Isotani, Ayako, Ikawa, Masahito
Format: Online
Language:English
Published: Nature Publishing Group 2013
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842082/
id pubmed-3842082
recordtype oai_dc
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 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/
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Mashiko, Daisuke
Fujihara, Yoshitaka
Satouh, Yuhkoh
Miyata, Haruhiko
Isotani, Ayako
Ikawa, Masahito
spellingShingle 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
author_facet Mashiko, Daisuke
Fujihara, Yoshitaka
Satouh, Yuhkoh
Miyata, Haruhiko
Isotani, Ayako
Ikawa, Masahito
author_sort Mashiko, Daisuke
title 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_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_sort generation of mutant mice by pronuclear injection of circular plasmid expressing cas9 and single guided rna
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.
publisher Nature Publishing Group
publishDate 2013
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842082/
_version_ 1612031231385403392

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