Identification of preexisting adaptive immunity to Cas9 proteins in humans

Abstract

The CRISPR–Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR–Cas9 system to therapeutically correct human genetic diseases1,2,3,4,5,6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR–Cas9 system moves toward clinical trials.

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Fig. 1: Identification of preexisting humoral immunity to Cas9.
Fig. 2: Identification of Cas9-specific T cell responses in humans.
Fig. 3: Isolation and restimulation of Cas9-specific T cells.

Data availability

The data supporting the findings of this study are available within the paper. Any additional data and materials that can be shared will be released via a Material Transfer Agreement.

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Acknowledgements

M.H.P. gratefully thanks the support of the Amon Carter Foundation and the Laurie Kraus Lacob Faculty Scholar Award in Pediatric Translational Research for this work. D.P.D. thanks the Stanford Child Health Research Institute Grant and Postdoctoral Award for supporting his work. C.T.C. was supported by the CIRM Bridges to Stem Cell Research Program (CIRM TB1-01190). We thank the Binns Family Program for Cord Blood Research (Stanford University) for providing the cord blood sera used in this work. We thank the other members of the Porteus laboratory for their helpful comments and suggestions.

Author information

N.G.-O. and M.P.-D. aided in the design of immunoblot assays to detect Cas9. C.T.C. performed all immunoblots. J.C., V.T.L., and B.J.L. designed and performed an ELISA assay to detect antibodies against Cas9 in human serum. C.A.V., M.A.C., N.M.B., L.Z., and M.A.B. cloned, purified, and tested for purity both homologs of Cas9 used throughout the study. P.S.D., B.D., and M.K.C. designed and implemented the T cell assays and B.D. designed the flow cytometry panels. R.R., C.T.C., and B.C. designed and implemented the T cell expansion assays and restimulation assays. S.M. provided the cord blood serum. M.K.C., J.C., and C.T.C. performed the Ficoll-Paque density gradient to purify human PBMC. M.P-D. and P.S.D designed the figures. M.K.C., N.G.-O., C.T.C, M.H.P., and P.S.D. wrote the manuscript. K.I.W., D.P.D., and M.H.P. directed the research and participated in the design and interpretation of the experiments.

Correspondence to Kenneth I. Weinberg or Matthew H. Porteus.

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Competing interests

M.H.P. serves on the Scientific Advisory Board and holds equity in CRISPR Therapeutics, but the company had no input into this work. C.A.V., M.A.C., L.Z., N.M.B., and M.A.B. are employees of Integrated DNA Technologies.

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Extended data

Extended Data Fig. 1 Initial screen for anti-Cas9 donors by immunoblot.

Immunoblot analysis using healthy donor serum to determine if humans have IgG antibodies to Cas9 proteins. Serum derived from cord blood (labeled ‘CB’) or adults (labeled ‘A’) and immunoreactivity against purified Cas9 proteins are shown. S.a, S. aureus Cas9 ortholog; S.p, S. pyogenes Cas9 ortholog. Each lane with a detectable band was analyzed for volume and the score of Cas9 to the background was measured by dividing the volume of the Cas9 band by the volume of the background. Each immunoblot was scored as either positive (+) or negative (−) for immunoreactivity. To be considered serum-positive for antibodies against either Cas9 ortholog, a band must have been present at the correct size on the immunoblot and have had a ratio of 1.10 over the background. A ratio of 1 indicates that no band could be detected at the correct molecular weight. Source Data

Extended Data Fig. 2 Titers of human serum concentrations used to detect anti-Cas9 antibodies with ELISA.

The same donor’s serum was applied at different concentrations by ELISA to detect antibodies against each antigen. The dilution of serum used to detect antibodies against each antigen is denoted above each graph.

Extended Data Fig. 3 Representative gating strategy to detect antigen-reactive T cells.

a, Representative gating strategy to detect antigen-reactive T cells by ICS. b, Representative gating strategy for detecting CD137 and CD154-positive T cells.

Extended Data Fig. 4 Cytokine responses to antigen stimulation of PBMCs.

a, Frequency of spots detected when 5 × 105 PBMCs were applied to wells of an ELISpot plate and challenged with each different antigen; the error bars represent the s.d. (n = 18). Significance was measured using a paired Student’s t-test. b, Frequency of T cells that were positive for different cytokines on antigen stimulation, as detected by ICS (n = 18). The black bars indicate the mean percentage of T cells positive for each cytokine (n = 18). *P < 0.05, **P < 0.01, ***P < 0.001, paired Student’s t-test. Each dataset was tested for significance against the unstimulated control. Source Data

Extended Data Fig. 5 FACS data from ICS and activation marker staining for donors 209 and 211–215.

Each individual donor number is shown at the top of each group of FACS plots. The red ‘+’ symbol below an antigen name indicates that a donor was considered positive for a cytokine response against that antigen by ICS. The red ‘−’ symbol below an antigen name indicates a donor was considered negative for a cytokine response to that antigen by ICS.

Extended Data Fig. 6 FACS data from ICS and activation marker staining for donors 16–221.

Each individual donor number is shown at the top of each group of FACS plots. The red ‘+’ symbol below an antigen name indicates that a donor was considered positive for a cytokine response against that antigen by ICS. The red ‘−’ symbol below an antigen name indicates that a donor was considered negative for a cytokine response to that antigen by ICS.

Extended Data Fig. 7 FACS data from ICS and activation markers staining for donors 223–228.

Each individual donor number is shown at the top of each group of FACS plots. The red ‘+’ symbol below an antigen name indicates that a donor was considered positive for a cytokine response against that antigen by ICS. The red ‘−’ symbol below an antigen name indicates that a donor was considered negative for a cytokine response to that antigen by ICS.

Extended Data Fig. 8 Results from expansion and restimulation of antigen-specific T cells against Cas9.

a, Results from the isolation, expansion, and restimulation of antigen-specific T cells against Cas9 from two additional PBMC donors to the donor presented in Fig. 3. b, Results from a technical replicate of expansion/restimulation of antigen-specific T cells against Cas9 from donor 213; the other replicate was presented in Fig. 3b.

Supplementary information

Reporting Summary

Supplementary Information

Supplementary Tables 1–4

Source data

Source data Fig. 1

Unprocessed Immunoblots

Source data Fig. 1

Statistical Source Data for ELISA

Source data Fig. 2

Statistical source data for intracellular cytokine staining

Source data Fig. 2

Statistical source data for Activation Markers

Source data Fig. 2

Statistical source data from ELISPOT

Source data Extended Data Fig. 1

Unprocessed immunoblots

Source data Extended Data Fig. 4

Statistical source data for intracellular cytokine staining

Source data Extended Data Fig. 4

Statistical source data from ELISPOT

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