文章基本信息

标题：Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA
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作者：Daisuke Mashiko ; Yoshitaka Fujihara ; Yuhkoh Satouh 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2013
卷号：3
期号：1
DOI：10.1038/srep03355
语种：English
出版社：Springer Nature
摘要：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.