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Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
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Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
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Journal Article
Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
2013
Overview
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 UK,Nature Publishing Group
Subject
/ 42/41
/ 42/70
/ 64/110
/ Animals
/ Calcium-Binding Proteins - genetics
/ Cellular Reprogramming Techniques - methods
/ Chromosomal Proteins, Non-Histone - genetics
/ Clustered Regularly Interspaced Short Palindromic Repeats - genetics
/ CRISPR
/ Genetic Engineering - methods
/ Genomes
/ Humanities and Social Sciences
/ Humans
/ Mice
/ Mutants
/ Mutation
/ Plasmids
/ RNA, Guide, CRISPR-Cas Systems
/ Rodents
/ Science
