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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.
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“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]
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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
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