Abstract
Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD-Rag1-/-IL2RgammaC-null (NRG) or NOD-SCID-IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF, and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.