Gene Targeting in Mouse Embryonic Stem Cells via CRISPR/Cas9 Ribonucleoprotein (RNP)-Mediated Genome Editing

The CRISPR/Cas9-mediated genome-editing system enables the development of gene-modified mice using fertilized eggs. However, while the efficiency in developing gene knockout mice by inducing small indel mutations would be good enough, the successful ratio to create large side DNA knock-in (KI) by embryonic genome editing is still low. In contrast to the direct embryo KI method, gene targeting using embryonic stem cells (ESC) followed by chimeric mouse development by blastocyst injection still has several advantages, e.g., high-throughput in vitro targeting/screening or large-size DNA KI such as Cre, CreERT, TetON, and reporter fluorescent protein, or their fusion proteins can be carried out without serving animal lives. The ESC targeting can also be applied to strains such as BALB/c, of which embryos are known to be difficult to handle in vitro. This text describes the optimized method for either short- or large-size DNA KI in ESC by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.