The DOI matches its Crossref record; 48 of the 48 references it cites were found in a registry or an index.
Identification of preexisting adaptive immunity to Cas9 proteins in humans
Open accessPublished version of a preprint
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 diseases 1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes 5,7. Given that these two bacterial species infect the human population at high frequencies 8,9, 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.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Second, long-term expression of genome editing molecules may expose patients to undesired off-target editing or immune reactions 66,67 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [66]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Pre-existing Cas9 antibodies and reactive T-cells have been detected in humans exposed to pathogenic bacteria harboring CRISPR systems, although it is unknown whether these are present at concentrations sufficient to trigger an immune response to the genome editing enzymes 66,104 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [66]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“although it is unknown whether these are present at concentrations sufficient to trigger an immune response to the genome editing enzymes 66,104 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [66]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Other factors affecting clinical applications of genome editing include the immunogenicity of bacterially-derived editing proteins, the potential for pre-existing antibodies against CRISPR components to cause inflammation and the unknown long-term safety and stability of genome editing outcomes.”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [66]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“Another consideration for CRISPR/Cas9-based systems for retinal gene editing is the potential for a heightened adaptive immune response given the bacterial origin of the Cas9 enzyme and high prevalence of pre-existing immunity (Charlesworth et al., 2019).”
From Gene Therapy-Associated Uveitis (GTAU): Understanding and mitigating the adverse immune response in retinal gene therapy (2025) · cites this paper as Charlesworth et al., 2019
In the citing paper
“A study involving 125 healthy adult human blood donors in the USA with a median age of 43 reported pre-existing anti-SaCas9 antibodies and anti-SpCas9 antibodies in 78% and 58% of the total samples, respectively.”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [24]
In the citing paper
“Among the tested donors, 78% had SaCas9-specific T-cells and 67% were positive for SpCas9-specific T-cells.”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [24]
In the citing paper
“While there are discrepancies regarding the exact prevalence of these antibodies in humans, a majority of the general population may exhibit preexisting α-Cas93.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [3]
In the citing paper
“Furthermore, a high prevalence of preexisting cellular immunity against Cas9 (Cas9-reactive T-cells) has also been identified2–4.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [3]
In the citing paper
“multiple groups have reported high frequencies (>60% of patients) of Cas9 reactive T-cells in humans2–4.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [3]
In the citing paper
“This may explain the lower raw ELISA A450 values we observed compared with previous work (Table 1)3.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [3]
In the citing paper
“CRISPR-Cas9 is a promising therapeutic tool that allows genetic target-specific cleavage and editing (111).”
From Immunomodulation in Administration of RAAV: Preclinical and Clinical Adjuvant Pharmacotherapies (2021) · cites this paper as [111]
In the citing paper
“One concern is that the prevalence of anti-SaCas9 antibodies and T-cell in humans are reported to be 78% (111).”
From Immunomodulation in Administration of RAAV: Preclinical and Clinical Adjuvant Pharmacotherapies (2021) · cites this paper as [111]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
Staphylococcus aureus Infections
Lowy, F. D. (1998) · New England Journal of Medicine · doi:10.1056/nejm199808203390806 · Cited by 6,018
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687
Manno, C. S., Pierce, G. F., Arruda, V. R., Glader, B., Ragni, M., Rasko, J. J. E., Ozelo, M. C., Hoots, K., Blatt, P., Konkle, B., et al. (2006) · Nature Medicine · doi:10.1038/nm1358 · Cited by 2,163
Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection
Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der Stegen, S. J. C., Hamieh, M., Cunanan, K. M., Odak, A., Gönen, M., & Sadelain, M. (2017) · Nature · doi:10.1038/nature21405 · Cited by 1,831
Zuris, J. A., Thompson, D. B., Shu, Y., Guilinger, J. P., Bessen, J. L., Hu, J. H., Maeder, M. L., Joung, J. K., Chen, Z.-Y., & Liu, D. R. (2015) · Nature Biotechnology · doi:10.1038/nbt.3081 · Cited by 1,460
In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration
Suzuki, K., Tsunekawa, Y., Hernandez-Benitez, R., Wu, J., Zhu, J., Kim, E. J., Hatanaka, F., Yamamoto, M., Araoka, T., Li, Z., et al. (2016) · Nature · doi:10.1038/nature20565 · Cited by 1,150
In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
Nelson, C. E., Hakim, C. H., Ousterout, D. G., Thakore, P. I., Moreb, E. A., Rivera, R. M. C., Madhavan, S., Pan, X., Ran, F. A., Yan, W. X., et al. (2016) · Science · doi:10.1126/science.aad5143 · Cited by 1,144
Pukelsheim, F. (1994) · The American Statistician · doi:10.1080/00031305.1994.10476030 · Cited by 1,072
Czerkinsky, C. C., Nilsson, L., Nygren, H., Ouchterlony, Ö., & Tarkowski, A. (1983) · Journal of Immunological Methods · doi:10.1016/0022-1759(83)90308-3 · Cited by 1,056
In vivo gene editing in dystrophic mouse muscle and muscle stem cells
Tabebordbar, M., Zhu, K., Cheng, J. K. W., Chew, W. L., Widrick, J. J., Yan, W. X., Maesner, C., Wu, E. Y., Xiao, R., Ran, F. A., et al. (2016) · Science · doi:10.1126/science.aad5177 · Cited by 1,046
Immune responses to AAV vectors: overcoming barriers to successful gene therapy
Mingozzi, F., & High, K. A. (2013) · Blood · doi:10.1182/blood-2013-01-306647 · Cited by 955
Finn, J. D., Smith, A. R., Patel, M. C., Shaw, L., Youniss, M. R., van Heteren, J., Dirstine, T., Ciullo, C., Lescarbeau, R., Seitzer, J., et al. (2018) · Cell Reports · doi:10.1016/j.celrep.2018.02.014 · Cited by 878
Yin, H., Song, C.-Q., Dorkin, J. R., Zhu, L. J., Li, Y., Wu, Q., Park, A., Yang, J., Suresh, S., Bizhanova, A., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3471 · Cited by 876
CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells
Dever, D. P., Bak, R. O., Reinisch, A., Camarena, J., Washington, G., Nicolas, C. E., Pavel-Dinu, M., Saxena, N., Wilkens, A. B., Mantri, S., et al. (2016) · Nature · doi:10.1038/nature20134 · Cited by 864
Self-citationWang, G., McCain, M. L., Yang, L., He, A., Pasqualini, F. S., Agarwal, A., Yuan, H., Jiang, D., Zhang, D., Zangi, L., et al. (2014) · Nature Medicine · doi:10.1038/nm.3545 · Cited by 837
Cd8(+) T-Cell Responses to Adeno-Associated Virus Capsid in Humans
Mingozzi, F., Maus, M. V., Hui, D. J., Sabatino, D. E., Murphy, S. L., Rasko, J. E. J., Ragni, M. V., Manno, C. S., Sommer, J., Jiang, H., et al. (2007) · Nature Medicine · doi:10.1038/nm1549 · Cited by 748
Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA
Long, C., McAnally, J. R., Shelton, J. M., Mireault, A. A., Bassel-Duby, R., & Olson, E. N. (2014) · Science · doi:10.1126/science.1254445 · Cited by 720
Dystrophin Immunity in Duchenne's Muscular Dystrophy
Mendell, J. R., Campbell, K., Rodino-Klapac, L., Sahenk, Z., Shilling, C., Lewis, S., Bowles, D., Gray, S., Li, C., Galloway, G., et al. (2010) · New England Journal of Medicine · doi:10.1056/nejmoa1000228 · Cited by 655
A Multifunctional AAV-CRISPR-Cas9 and Its Host Response
Chew, W. L., Tabebordbar, M., Cheng, J. K. W., Mali, P., Wu, E. Y., Ng, A. H. M., Zhu, K., Wagers, A. J., & Church, G. M. (2016) · Nature Methods · doi:10.1038/nmeth.3993 · Cited by 617
The MHC class I antigen presentation pathway: strategies for viral immune evasion
Hewitt, E. W. (2003) · Immunology · doi:10.1046/j.1365-2567.2003.01738.x · Cited by 491
DeWitt, M. A., Magis, W., Bray, N. L., Wang, T., Berman, J. R., Urbinati, F., Heo, S.-J., Mitros, T., Muñoz, D. P., Boffelli, D., et al. (2016) · Science Translational Medicine · doi:10.1126/scitranslmed.aaf9336 · Cited by 477
Direct access to CD4+ T cells specific for defined antigens according to CD154 expression
Frentsch, M., Arbach, O., Kirchhoff, D., Moewes, B., Worm, M., Rothe, M., Scheffold, A., & Thiel, A. (2005) · Nature Medicine · doi:10.1038/nm1292 · Cited by 475
High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population
Wagner, D. L., Amini, L., Wendering, D. J., Burkhardt, L.-M., Akyüz, L., Reinke, P., Volk, H.-D., & Schmueck-Henneresse, M. (2019) · Nature Medicine · doi:10.1038/s41591-018-0204-6 · Cited by 399
Wang, D., Mou, H., Li, S., Li, Y., Hough, S., Tran, K., Li, J., Yin, H., Anderson, D. G., Sontheimer, E. J., et al. (2015) · Human Gene Therapy · doi:10.1089/hum.2015.087 · Cited by 345
Lessons learned from the gene therapy trial for ornithine transcarbamylase deficiency
Wilson, J. M. (2009) · Molecular Genetics and Metabolism · doi:10.1016/j.ymgme.2008.12.016 · Cited by 299
Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo
Yang, Y., & Wilson, J. M. (1995) · The Journal of Immunology · doi:10.4049/jimmunol.155.5.2564 · Cited by 262
Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population
Simhadri, V. L., McGill, J., McMahon, S., Wang, J., Jiang, H., & Sauna, Z. E. (2018) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2018.06.006 · Cited by 241
VSV-G Pseudotyped Lentiviral Vector Particles Produced in Human Cells Are Inactivated by Human Serum
DePolo, N. J., Reed, J. D., Sheridan, P. L., Townsend, K., Sauter, S. L., Jolly, D. J., & Dubensky, T. W. (2000) · Molecular Therapy · doi:10.1006/mthe.2000.0116 · Cited by 223
Simms, P. E., & Ellis, T. (1996) · Clinical and Diagnostic Laboratory Immunology · doi:10.1128/cdli.3.3.301-304.1996 · Cited by 208
Smith, K. M., Pottage, L., Thomas, E. R., Leishman, A. J., Doig, T. N., Xu, D., Liew, F. Y., & Garside, P. (2000) · The Journal of Immunology · doi:10.4049/jimmunol.165.6.3136 · Cited by 185
Dryla, A., Prustomersky, S., Gelbmann, D., Hanner, M., Bettinger, E., Kocsis, B., Kustos, T., Henics, T., Meinke, A. L., & Nagy, E. (2005) · Clinical and Vaccine Immunology · doi:10.1128/cdli.12.3.387-398.2005 · Cited by 183
Bacher, P., Schink, C., Teutschbein, J., Kniemeyer, O., Assenmacher, M., Brakhage, A. A., & Scheffold, A. (2013) · The Journal of Immunology · doi:10.4049/jimmunol.1202221 · Cited by 173
Single step protocol to purify recombinant proteins with low endotoxin contents
Reichelt, P., Schwarz, C., & Donzeau, M. (2005) · Protein Expression and Purification · doi:10.1016/j.pep.2005.09.027 · Cited by 166
Roberts, A. L., Connolly, K. L., Kirse, D. J., Evans, A. K., Poehling, K. A., Peters, T. R., & Reid, S. D. (2012) · BMC Pediatrics · doi:10.1186/1471-2431-12-3 · Cited by 139
The Fall of a Dogma? Unexpected High T-Cell Memory Response toStaphylococcus aureusin Humans
Kolata, J., Kühbandner, I., Link, C., Normann, N., Vu, C. H., Steil, L., Weidenmaier, C., & Bröker, B. M. (2015) · The Journal of Infectious Diseases · doi:10.1093/infdis/jiv128 · Cited by 121
Multiparameter Intracellular Cytokine Staining
Lovelace, P., & Maecker, H. T. (2010) · Methods in molecular biology · doi:10.1007/978-1-61737-950-5_8 · Cited by 85
Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans
Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Dejene, B., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Camarena, J., Weinberg, K. I., & Porteus, M. H. (2018) · doi:10.1101/243345 · Cited by 71
Self-citationLevels of Antibody against 11 Staphylococcus aureus Antigens in a Healthy Population
Colque‐Navarro, P., Jacobsson, G., Andersson, R., Flock, J., & Möllby, R. (2010) · Clinical and Vaccine Immunology · doi:10.1128/cvi.00506-09 · Cited by 70
Mortensen, R., Nissen, T. N., Blauenfeldt, T., Christensen, J. P., Andersen, P., & Dietrich, J. (2015) · The Journal of Immunology · doi:10.4049/jimmunol.1500804 · Cited by 55
Ford, T., Wenden, C., Mbekeani, A. J., Dally, L., Cox, J. H., Morin, M. J., Winstone, N., Hill, A. V. S., Gilmour, J. W., & Ewer, K. (2017) · Vaccine · doi:10.1016/j.vaccine.2017.02.038 · Cited by 48
T cell activation: in vivo veritas
Groth, B. F. D. S., Smith, A. L., & Higgins, C. A. (2004) · Immunology and Cell Biology · doi:10.1111/j.0818-9641.2004.01243.x · Cited by 42
Christmas, S. E., & Meager, A. (1990) · PubMed Central · Cited by 39
Rapid detection, enrichment and propagation of specific T cell subsets based on cytokine secretion
Campbell, J. D. M., Foerster, A. M. H., Lasmanowicz, V., Niemöller, M., Scheffold, A., Fahrendorff, M., Rauser, G., Assenmacher, M., & Richter, A. (2010) · Clinical & Experimental Immunology · doi:10.1111/j.1365-2249.2010.04261.x · Cited by 39
Antiviral Effect of TNF-α and IFN-γ Secreted from a CD8 + Influenza Virus-Specific CTL Clone
Kuwano, K., Kawashima, T., & Arai, S. (1993) · Viral Immunology · doi:10.1089/vim.1993.6.1 · Cited by 27
Möller, J. F., Möller, B., Wiedenmann, B. J., Berg, T., & Schott, E. (2011) · Journal of Viral Hepatitis · doi:10.1111/j.1365-2893.2010.01430.x · Cited by 22
Gupta, S., & Maecker, H. T. (2015) · BIO-PROTOCOL · doi:10.21769/bioprotoc.1442 · Cited by 7
Cited by
Sorted by citations
Car t cell immunotherapy for human cancer
June, C. H., O’Connor, R. S., Kawalekar, O. U., Ghassemi, S., & Milone, M. C. (2018) · Science · doi:10.1126/science.aar6711 · Cited by 3,207
Li, H., Yang, Y., Hong, W., Huang, M., Wu, M., & Zhao, X. (2020) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-019-0089-y · Cited by 1,829
The CRISPR tool kit for genome editing and beyond
Adli, M. (2018) · Nature Communications · doi:10.1038/s41467-018-04252-2 · Cited by 1,545
CRISPR-engineered T cells in patients with refractory cancer
Stadtmauer, E. A., Fraietta, J. A., Davis, M. M., Cohen, A. D., Weber, K. L., Lancaster, E., Mangan, P. A., Kulikovskaya, I., Gupta, M., Chen, F., et al. (2020) · Science · doi:10.1126/science.aba7365 · Cited by 1,478
The next generation of CRISPR–Cas technologies and applications
Pickar-Oliver, A., & Gersbach, C. A. (2019) · Nature Reviews Molecular Cell Biology · doi:10.1038/s41580-019-0131-5 · Cited by 1,459
Metadata
- Authors
- Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., Behlke, M. A., Dejene, B., Cieniewicz, B., Romano, R., Lesch, B. J., Gomez-Ospina, N., Mantri, S., Pavel-Dinu, M., Weinberg, K. I., & Porteus, M. H.
- Affiliations
- Stanford Medicine (US)
- Integrated DNA Technologies (United States) (US)
- Corresponding author
- Weinberg, Kenneth I.
- Porteus, Matthew H.
- ORCID
- Charlesworth, Carsten T.
- Dever, Daniel P.
- Camarena, Joab
- Lemgart, Viktor T.
- Cromer, M. Kyle
- Vakulskas, Christopher A.
- Collingwood, Michael A.
- Zhang, Liyang
- Bode, Nicole M.
- Behlke, Mark A.
- Dejene, Beruh
- Cieniewicz, Brandon
- Romano, Rosa
- Lesch, Benjamin J.
- Gomez-Ospina, Natalia
- Mantri, Sruthi
- Pavel-Dinu, Mara
- Weinberg, Kenneth I.
- Porteus, Matthew H.
- Venue
- Nature Medicine
- Published
- 2019-01-28
- Volume
- 25
- Issue
- 2
- Pages
- 249-254
- ISSN
- 1078-8956 (ISSN-L)
- 1546-170X
- PMC
- PMC7199589
- OpenAlex
- W2949380898
- Cited by
- 1,093
- References
- 48
- Citations by year
- 2026: 130 · 2025: 128 · 2024: 132 · 2023: 111 · 2022: 125 · 2021: 166 · 2020: 131 · 2019: 110
- FWCI
- 44.16
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Medicine (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by-nd)
- Funders
- Amon G. Carter Foundation
- National Institutes of Health (5r01ai120766-02)
- National Institute of General Medical Sciences (T32GM119995)
- National Institute of Allergy and Infectious Diseases (R01-AI120766)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, SaCas9, SpCas9, Staphylococcus aureus, Streptococcus pyogenes, humoral immune response, cell-mediated immunity, genome editing safety
- MeSH terms
- CRISPR-Associated Protein 9, Adult, Cell Separation, Female, Humans, Male, T-Lymphocytes, Adaptive Immunity, Immunity, Humoral
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
