The discovery of the highly efficient site-specific nuclease system CRISPR–Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy^,. The immunogenicity of Cas9 nuclease has been demonstrated in mice^,. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues^{, , –}. 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 nuclease^,. 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.