The discovery of the highly efficient site-specific nuclease system CRISPR-Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy1,2. The immunogenicity of Cas9 nuclease has been demonstrated in mice3,4. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues3-6. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease7,8. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell-mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.