Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.