Identification of preexisting adaptive immunity to Cas9 proteins in humans

The CRISPR–Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR–Cas9 system to therapeutically correct human genetic diseases 1 – 6 . The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes 5 , 7 . Given that these two bacterial species infect the human population at high frequencies 8 , 9 , we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 ( S. aureus ) and SpCas9 ( S. pyogenes ). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR–Cas9 system moves toward clinical trials.