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Updated: Jan 25, 2026
Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
Published on: November 3, 2020
Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes
Shayesteh R Ferdosi1,2, Radwa Ewaisha1,3, Farzaneh Moghadam4
1Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA.
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.
Insights
Pre-existing immunity to CRISPR-Cas9 (SpCas9) is common in healthy individuals, posing challenges for gene therapies. This study identifies key immune epitopes and demonstrates a method to modify SpCas9 to reduce immune responses.
Area of Science:
- Immunology
- Gene Therapy
- Molecular Biology
Background:
- CRISPR-Cas9 gene editing holds promise for personalized therapies.
- The bacterial Cas9 protein's immunogenicity is a significant concern for human applications.
- Pre-existing immunity can limit the efficacy of CRISPR-based treatments.
Purpose of the Study:
- To investigate pre-existing immunity to Streptococcus pyogenes Cas9 (SpCas9) in healthy individuals.
- To identify immunodominant T cell epitopes within SpCas9.
- To develop strategies for mitigating SpCas9 immunogenicity in gene therapy.
Main Methods:
- Serological screening for antibodies against SpCas9.
- T cell assays using peripheral blood mononuclear cells (PBMCs) from healthy donors.
- Epitope prediction algorithms incorporating T cell receptor contact residue hydrophobicity and HLA binding.
- Functional and specificity assays of modified SpCas9 variants.
Main Results:
- Antibodies to SpCas9 were detected in at least 5% of 143 healthy individuals.
- The majority of screened healthy individuals exhibited pre-existing CD8+ T cell immunity to SpCas9.
- Two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 were identified.
- Cas9 protein was successfully modified to eliminate identified epitopes while retaining function and specificity.
Conclusions:
- Pre-existing immunity to CRISPR-associated nucleases like SpCas9 is prevalent.
- Targeted modification of SpCas9 can reduce T cell immunogenicity.
- This approach offers a potential strategy to overcome immune barriers in CRISPR-based gene therapies.
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