Fig. 1
From: Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes
Detection of pre-existing B cell and T cell immune responses to SpCas9 in healthy donors and identification of two immunodominant T cell epitopes. a Specific serum Abs were detected against S. pyogenes lysate in 57.3% (n = 82) of 143 healthy controls. Sera with the highest reactivity to S. pyogenes lysate (n = 80, black circles) were screened for Abs against recombinant SpCas9, recombinant EBNA-1 protein (positive control), and human hemoglobin (negative control), of which 7 (8.8%) were positive for SpCas9 (above the dotted line; *p < 0.0001). b The top 5 predicted SpCas9 T cell epitopes and their predicted Sb and Si scores and ranking (based on the Sb.Si value)32. These top 5 peptides include the identified immunodominant (α and β; gray) and subdominant (γ and δ) epitopes that were shown to be immunogenic by IFN-γ ELISpot. c Plot of Sb and Si of predicted HLA-A*02:01 epitopes for the SpCas9 protein. Red dots represent the immunodominant and subdominant epitopes. d IFN-γ ELISpot assay of T cell reactivity of 12 healthy donors (the two non- HLA-A*02:01 are shown as open circles) to 38 predicted epitopes grouped in 10 pools, CEF (positive control peptide pool), and DMSO (negative control). Peptides α and δ were in pool 5 while β and γ were in pool 3. e IFN-γ ELISpot reactivity of healthy donor T cells (n = 12) to epitopes across the different domains of the Cas9 protein. Donors 1–10 were HLA-A*02:01, while 11 and 12 were not. Peptides α and δ overlap in 5 amino acid residues. Data represent mean ± SD. EBNA-1, Epstein-Barr virus nuclear antigen-1; Sb, normalized binding score; Si, normalized immunogenicity score. Statistical analysis was performed post hoc and results are exploratory. Source data are available in the Source Data file
