State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology, Ministry of Education, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
CRISPR J. 2022 Jun;5(3):445-456. doi: 10.1089/crispr.2021.0142.
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 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-A24:02, HLA-A11:01, 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.
CRISPR-Cas9 系统正越来越多地被用作复杂疾病的基因编辑治疗技术，但人们仍然对 Cas9 免疫原性的临床风险感到担忧。在这项研究中，我们分别在 4.8%和 70%的中国供体中检测到针对 Cas9（SaCas9）的抗体和抗 SaCas9 T 细胞。我们预测了针对 HLA-A24:02、HLA-A11:01 和 HLA-A*02:01 的 135 个 SaCas9 衍生 B 细胞表位和 50 个 SaCas9 衍生 CD8+ T 细胞表位。通过使用针对 SaCas9 特异性抗体呈阳性的血清和针对 SaCas9 反应性 T 细胞呈阳性的外周血单核细胞进行免疫测定，我们分别鉴定出 R338 是三种人类白细胞抗原同种型的免疫显性 SaCas9 B 细胞表位，SaCas9_200-208 是免疫显性 CD8+ T 细胞表位。我们还证明，携带 R338G 取代的 SaCas9 变体降低了 B 细胞免疫原性，但保留了其基因编辑功能。我们的研究强调了 CRISPR-Cas9 系统的免疫风险，并提供了一种解决方案，可以减轻人类对 Cas9 预先存在的适应性免疫反应。
