Unwanted alloimmune responses are a central driver of solid organ transplant rejection and currently managed with life-long immunosuppression, which imposes substantial risks and burdens on patients. Adoptive transfer of regulatory T cells (Tregs) offers a strategy to restore immunological balance and reduce long-term adverse effects of generalized immunosuppression. However, although Tregs can potently inhibit incipient immune activation, they struggle to suppress established memory effector T cells, necessitating the continued use of immunosuppression. Calcineurin inhibitors such as Tacrolimus effectively control both, newly activated and pre-existing effector T cells, but unfortunately, they also impair Treg function. Therefore, we hypothesized that gene-editing of Tregs inducing tacrolimus resistance (FKBP12KO) would enable combined therapy that curbs effector T cell responses without compromising Treg efficacy. Here, we developed FKBP12KO-Tregs using a ribonucleoprotein-based CRISPR-Cas9 approach and characterized them extensively in vitro. FKBP12KO-Tregs retained phenotype, high viability, and suppressive function comparable to unedited TregWT, and they remained functionally impervious to Tacrolimus, while preserving sensitivity to alternative CNIs. We additionally established a good-manufacturing practice process for FKBP12KO-Tregs. Comprehensive in vitro phenotypic, functional, and molecular characterization, together with the established manufacturing, provide the rationale for a proof-of-concept clinical trial assessing the feasibility and safety of co-administration of FKBP12KO-Tregs with Tacrolimus in living-donor kidney transplant recipients.