Very good paper with detailed protocols -
Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.
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