CRISPR/Cas9 DNA synthesis disruption as a tool to control cell proliferation in vitro and in vivo

Cell therapies hold a lot of promise for regenerative medicine and gene therapy. However, many living cell products entail a fundamental biological risk of unwanted growth. Here, we describe a novel metabolic safety system to control cell proliferation without exogenous genetic elements. The method allows robust cell culture and manufacture while mitigating the risk of uncontrolled growth of transplanted cells. Using CRISPR/Cas9, we inactivated the enzyme in charge of the de novo thymidylate synthesis (TYMS) in several cell lines, including human induced pluripotent stem cells. This resulted in cells that proliferate when supplemented with exogenous dTMP but fail to grow in its absence. Under dTMP supplementation, TYMS-/- hiPSCs produce mature teratomas in vivo and differentiate normally into potentially therapeutic cell types, such us pancreatic beta cells. After terminal differentiation, the cells no longer require dTMP to remain functional, as seen by prolonged in vivo production of human insulin in mice. ### Competing Interest Statement The authors have declared no competing interest.