Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes

Abstract

The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.

Fig. 1

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 S_b and S_i scores and ranking (based on the S_b.S_i value). 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 S_b and S_i 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; S_b, normalized binding score; S_i, normalized immunogenicity score. Statistical analysis was performed post hoc and results are exploratory. Source data are available in the Source Data file

Fig. 2

SpCas9 immunodominant epitope-specific CD8+T cell recognition is abolished after anchor residue mutation. a Epitope β-specific CD8+T cell response detected using β-specific pentamer in PBMCs stimulated with peptide β-pulsed antigen presenting cells. b The percentage of CD8+ pentamerβ+T cells was reduced to 0.3% when healthy donor B cell APCs were pulsed with the mutated peptide β2. c Positions, sequences, and IEDB HLA binding percentile rank of epitopes α and β before and after mutation of the anchor (2nd and/or 9th) residues. S_b, normalized binding score; S_i, normalized immunogenicity score. d Representative IFN-γ ELISpot assay in triplicate wells comparing T cell reactivity to wild type or mutated epitopes α and β. These results are representative of 12 donors and two independent replicates (data from all 12 donors are shown in Supplementary Fig. 1). e, f IFN-γ ELISpot comparing T cell reactivity to APCs expressing WT or modified Cas9 proteins. APCs expressing FluM1 were used as a positive control. APCs expressing GAPDH or spiked with peptide α2 were used as negative controls. Data represent mean ± SEM of 5 replicates (right). Statistical analysis was performed post hoc and results are exploratory. Source data are available in the Source Data file

Fig. 3

Mutated SpCas9 protein (Cas9-β2) retains its function and specificity. a 3D structure of the SpCas9 protein, showing the location of the identified immunodominant epitopes α and β. b Schematic of the experiment assessing mutagenesis capacity of Cas9-β2. Cells were transfected with either WT-Cas9, Cas9-β2, or an empty plasmid as well as 20 nt gRNA targeting EMX-1 locus. 72 h after transfection, percent cleavage was assessed by DNA extraction and illumina sequencing. c Percentage of indel formation in EMX-1 locus. Data represent mean ± SD of three individual transfections. d Schematic of the experiment assessing gRNA binding, DNA targeting and transcriptional modulation with Cas9-β2. Cells were transfected with either WT-Cas9, Cas9-β2, or an empty plasmid as well as 14nt gRNA targeting TTN or MIAT in the presence of MS2-P65-HSF1 (transcriptional modulation). 72 h after transfection, mRNA was assessed by qRT-PCR. e, f Shown is the mRNA level relative to an untransfected control experiment (n = 3 independent technical replicaes). g Mean expression levels of 24,078 protein-coding and non-coding RNA genes for WT-Cas9 and Cas9-β2 (each in duplicate) are shown. For visualization purposes, the values were transformed to a log₂(CPM+1) scale. MIAT, the gRNA target gene, is highlighted in red, and R denotes Pearson correlation coefficient between two groups. Source data are available in the Source Data file

Fig. 4

Immune responses to non-HLA-A*02:01 and class II epitopes of SpCas9. a Representative IFN-γ ELISpot reactivity of healthy donor T cells to non-HLA-A*02:01 SpCas9 class I epitopes (HLA-A*024:02 is shown as an example). b IFN-γ ELISpot reactivity of healthy donor MACS-sorted CD4⁺ T cells to SpCas9 long peptides that include epitopes in the top 2% of predicted MHC class II binders. c IFN-γ ELISpot reactivity of healthy donor CD8-depleted PBMCs stimulated with recombinant SpCas9 or EBNA proteins for 10 days. d IFN-γ ELISpot reactivity of MACS sorted CD4⁺ (black dots) or CD8⁺ (open dots) T cells isolated from PBMCs from three healthy donors unstimulated or stimulated with peptide β or CEF. Data represent mean ± SD. Source data are available in the Source Data file