PROJECT SUMMARY: Genetically modified (GM) animals are essential tools for the study of both fundamental biology and human diseases. The production of GM animals relies on two critical technologies: 1) stable genetic modifications and 2) germline transmission of the mutations into a model system. A typical approach for creation of complex GM mice involves the generation of tetra-parental chimeras from normal embryos and GM embryonic stem (ES) cells, followed by multiple rounds of breeding to obtain both male and female mice for germline propagation. Two limitations dominate this approach. First, maintenance of pluripotency limits the complexity of genetic manipulations. Second, this process is time-consuming, laborious, and costly, particularly if the final objective requires many independent germline manipulations in the same animal. We propose a feasible strategy to accelerate the production of complex GM mouse models. Employing the technology of sex-reversion via CRISPR/Cas9-meditated Y chromosome deletion in male ES cells and our novel ES cell culture system, we can directly generate isogenic male and female mice from the same targeted ES cells through tetraploid complementation (4n). This strategy would bypass at least two mouse breeding generations: the chimera development step and the complex breeding process. We will target male (XY) ES cell lines for intended genetic alterations and follow the deletion of Y chromosome to generate monosomic XO female ES cells. Using this strategy, compound homozygous GM mouse strains could be established at unparalleled speed and costs. In this R21 application we propose the following two Aims: Aim 1: Optimize AX-based ES cell culture system for the production of GM mouse models. Aim 2. Direct generation of isogenic male and female complex GM mice using novel ES cell culture medium. If successful, our approach would have a great impact on GM mouse model construction in terms of versatility, speed, and cost. This ambitious endeavor to develop a breakthrough technology for creation of complex GM mouse models would also possibly foster novel research opportunities.