RNA-guided RNA-targeting nucleases, such as CRISPR–Cas13 proteins, have therapeutic potential for gene editing. Among Cas13d enzymes, Cas13d from the bacteria Ruminococcus flavefaciens (RfxCas13d) is of particular interest owing to its small size and high specificity. However, the existence of pre-existing immunity against RfxCas13d is unclear. In this study, we evaluated antibody and T cell responses to RfxCas13d in healthy donors using ELISA and T cell culture assays. We found RfxCas13d-reactive antibodies and CD4 and CD8 T cell responses in most donors, comparable to responses against Cas9 proteins from Staphylococcus aureus (SaCas9) and Streptococcus pyogenes (SpCas9). RfxCas13d-responding T cells could produce the inflammatory cytokines IFN-γ, TNF-α and IL-17. These findings should be taken into consideration in the development of RfxCas13d for therapy.

RNA 引导的 RNA 靶向核酸酶，如 CRISPR–Cas13 蛋白，具有基因编辑的治疗潜力。在 Cas13d 酶中，来自细菌 Ruminococcus flavefaciens ( RfxCas13d) 的 Cas13d 由于其小尺寸和高特异性而受到特别关注。然而，是否存在针对 RfxCas13d 的预先存在的免疫力尚不清楚。在这项研究中，我们使用 ELISA 和 T 细胞培养试验评估了健康供体中 RfxCas13d 的抗体和 T 细胞反应。我们在大多数供体中发现了 RfxCas13d 反应性抗体和 CD4 和 CD8 T 细胞反应，与对来自金黄色葡萄球菌(SaCas9) 和化脓性链球菌的 Cas9 蛋白的反应相当(SpCas9)。响应 RfxCas13d 的 T 细胞可以产生炎性细胞因子 IFN-γ、TNF-α 和 IL-17。在开发用于治疗的 RfxCas13d 时应考虑这些发现。