Engineering Immunodominant Residues of Cas9 reduces Cell Mediated Cytotoxicity

Identification and engineering of TCR epitopes in Cas9 protein helps reduce T cell response against CRISPR

CRISPR is a bacterial-derived gene editing tools that can potentially cure various genetic disorders. CRISPR consist of a Cas9 protein that creates a nick in the specific site of double-stranded DNA that is recognized by guide RNA. During the double strand break repairs, the target site may be engineered to obtain the intended outcomes. The adeno-associated viral vectors are most commonly used for delivery of gene and long term expression of Cas9 proteins. These endogenously synthesized Cas9 proteins are processed and presented via MHC-I in the target cells. Any preexisting reactive T cells recognize to MHC- Cas9 peptide complex and elicit cell-mediated cytotoxicity. This may have a detrimental effect on the tissues that express Cas9 transgene.

When T cell encounters the target cells, T cell receptor bind to the MHC class I presented with short antigenic peptides (8-10 amino acid residue). These peptides (T cell epitopes) consist of signature hydrophobic amino acids that interact with MHC-I and T cells. Identifying these T cell epitopes and engineering it to reduce its binding affinity towards MHC and T cell could reduce the T cell response against Cas9. Recently Ferdosi et al showed that CRISPR/Cas9 may be engineered to immunosilience human epitopes without altering DNA editing efficiency.


a) Identification of T cell epitopes
b) Evaluation of T cell response on mutant Cas9 protein
c) Evaluation of gene editing efficiency

a) Identification of T cell epitopes:
The T cell receptor interacts with signature hydrophobic amino acid residues present in T cell epitopes. The scanning of hydrophobic amino acid residues throughout the Cas9 and ranking short segments of peptides is the first step in the identification of the T cell epitopes. Ferdosi et al. used a previously published algorithm known as a hydrophobicity-based artificial neural network (ANN-Hydro) which utilizes T cell contact residue hydrophobicity & HLA binding prediction to determine the immunogenic epitopes. The short peptide identified were tested in vitro to determine the immunoreactivity using ELISPOT. The results showed the immunodominant residues are located in the REC lobe of the Cas9 protein.





b) Evaluation of T cell response on mutant Cas9 protein:
In theory, the modification of hydrophobic contact in the REC lobe of Cas9 protein should abolish T cell receptor recognition. To test this hypothesis, they created a mutant Cas9 lacking these residues and evaluated the T cell reactivity using IFN-γ ELISPOT assay and showed the marked reduction of T cell reactivity compared to wildtype.

c) Evaluation of gene editing efficiency:
To validate that the modification of hydrophobic contact residues does reduce the gene editing efficiency, authors designed three guide RNA that target different DNA constructs (as shown in the figure) and showed a minimal impact of mutant Cas9 on the editing efficiency compared to wildtype.

In conclusion, this study provides the proof-of-concept on how we can leverage the understanding of T cell biology and genetics to identify and engineering the immunodominant region of the peptide and reduce the immunogenicity.

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