Genetically Modified Mouse Models and Applications

“The generation of genetically modified mouse models has revolutionized our ability to study gene function and disease mechanisms. The CRISPR/Cas9 system, a precise and efficient genome-editing tool, has become a cornerstone for creating knockout (KO), conditional knockout (CKO), and knock-in (KI) mouse models. This system employs guide RNAs (gRNAs) to target specific genomic loci, enabling gene editing through double-strand breaks and subsequent DNA repair mechanisms.
In KO models, CRISPR/Cas9 introduces frameshift mutations through non-homologous end joining (NHEJ), effectively disrupting gene function. CKO models are generated by integrating loxP sites flanking critical exons, allowing tissue- or time-specific gene deletion using Cre recombinase. For KI models, homology-directed repair (HDR) facilitates the insertion of precise genetic modifications, such as point mutations or reporter genes. This approach ensures high specificity and versatility in recapitulating human genetic conditions.
Here, we outline protocols and considerations for CRISPR/Cas9-mediated mouse model generation, including gRNA design, delivery methods (e.g., microinjection or electroporation in zygotes), and validation strategies. We also address common challenges, such as off-target effects and mosaicism, and discuss recent advancements in improving efficiency and accuracy. These models provide invaluable tools for functional genomics, preclinical research, and therapeutic discovery.”