Rapid generation of conditional knockout mice using the CRISPR-CAS9 system and electroporation for neuroscience research

The Cre/Loxp-based conditional knockout technology is a powerful tool for gene function analyses by allowing region-time-specific gene manipulation. However, inserting a pair of LoxP cassettes for generating conditional knock-out can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using the in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exon 5 to exon 6 of CaMK1 in a short period, 125 days, using only 16 mice. The efficiency of generating floxed mice was 10%, significantly higher than the conventional ES cell-based method. We directly edited the genome of C57BL/6N fertilized eggs, our target genetic background, to eliminate additional backcrossing steps. We confirmed that the genome of this floxed mouse is responsive to Cre protein. This low-cost, highly efficient method for generating conditional knock-out will facilitate comprehensive, tissue-specific genome analyses.