Modern Approaches to Mouse Genome Editing Using the CRISPR-Cas Toolbox and Their Applications in Functional Genomics and Translational Research.

Mice have been used in biological research for over a century, and their immense contribution to scientific breakthroughs can be seen across all research disciplines, with some of the main beneficiaries being the fields of medicine and life sciences. Genetically engineered mouse models (GEMMs), along with other model organisms, are fundamentally important research tools frequently utilised to enhance our understanding of pathophysiology and biological mechanisms behind disease. In the 1980s, it became possible to precisely edit the mouse genome to create gene knockout and knock-in mice, although with low efficacy. Recent advances utilising CRISPR-Cas technologies have considerably improved our ability to do this with ease and precision, while also allowing the generation of desired genetic variants from single nucleotide substitutions to large insertions/deletions. It is now quick and relatively easy to genetically edit somatic cells which were previously more recalcitrant to traditional approaches. Further refinements have created a 'CRISPR toolkit' that has expanded the use of CRISPR-Cas beyond gene knock-ins and knockouts. In this chapter, we review some of the latest applications of CRISPR-Cas technologies in GEMMs, including nuclease-dead Cas9 systems for activation or repression of gene expression, base editing and prime editing. We also discuss improvements in Cas9 specificity, targeting efficacy and delivery methods in mice. Throughout, we provide examples wherein CRISPR-Cas technologies have been applied to target clinically relevant genes in preclinical GEMMs, both to generate humanised models and for experimental gene therapy research.