Transgenic lineage-tracing mice are among the most influential biomedical research technologies. Dual lineage-tracing mice provide further information on phenotypic switching of cell fates during normal development, organ function, and disease. This proposal seeks to generate a series of new unique dual lineage-tracing transgenic mice to investigate angiogenesis and fibrosis in multiple disease models. Cycling endothelial cell (EC) are required for angiogenesis and endothelial-to-mesenchymal transition (EndMT) participates in fibrosis of disease. However, delineating the contributions of adult ECs that reenter the cell cycle or undergo EndMT remains a challenge. Although current lineage-tracing mice label cells, these approaches cannot ablate specific subpopulations of ECs that cycle or undergo EndMT to determine their contributions to disease progression and severity. To address this knowledge gap, we will use our previously published methods of sequential orthologous DNA recombinases to fine-tune DNA recombination temporally in specific cells. Sequential DNA recombinases allow the ablation and interrogation of subsequent effects on organ function. Applying this strategy to cardiomyocytes (CMs), we created a new transgenic mouse that expressed tandem orthologous DNA recombinases. We observed that ablating endogenous cycling CMs worsened heart function after myocardial infarction (MI). We used conventional mouse transgenesis for the CM experiments, a laborious, expensive, and time- consuming process, taking over twelve months to obtain mice needed for investigations. However, advances in “Targeted Integration with Linearized dsDNA” (TILD) - “Clustered Regularly Interspaced Short Palindromic Repeats” (CRISPR) provide a remarkably accurate, efficient, and rapid alternative to generate transgenic mice. Therefore, we will merge CRISPR-mediated genome editing and orthologous DNA recombinases to create a toolbox of next-generation transgenic mice that can be “mixed-and- matched” to investigate EC cycling and EndMT-mediated fibrosis in vivo. The new mice will expand our knowledge of EC proliferation and EndMT-mediated fibrosis in multiple diseases, including cardiac fibrosis, atherosclerosis, pulmonary arterial hypertension, cirrhosis, and cancer. The methods developed will enable the rapid creation of various transgenic mice that use sequential DNA recombinases to restrict Cre expression and investigate cell cycling and cell fate switching in vivo.