The CRISPR-Cas9 system is increasingly being used as a gene editing therapeutic technique in complex diseases but concerns remain regarding the clinical risks of Cas9 immunogenicity. In this study, we detected antibodies against Staphylococcus aureus Cas9 (SaCas9) and anti-SaCas9 T cells in 4.8% and 70% of Chinese donors, respectively. We predicted 135 SaCas9-derived B cell epitopes and 50 SaCas9-derived CD8+ T cell epitopes for HLA-A*24:02, HLA-A*11:01, and HLA-A*02:01. We identified R338 as an immunodominant SaCas9 B cell epitope and SaCas9_200–208 as an immunodominant CD8+ T cell epitope for the three human leukocyte antigen allotypes through immunological assays using sera positive for SaCas9-specific antibodies and peripheral blood mononuclear cells positive for SaCas9-reactive T cells, respectively. We also demonstrated that an SaCas9 variant bearing an R338G substitution reduces B cell immunogenicity and retains its gene-editing function. Our study highlights the immunological risks of the CRISPR-Cas9 system and provides a solution to mitigate pre-existing adaptive immune responses against Cas9 in humans.
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