Targeted modification of major histocompatibility complex class 1 molecules via CRISPR-CAS9

Nguyen, Dinh Dung (2022) Targeted modification of major histocompatibility complex class 1 molecules via CRISPR-CAS9. Doctoral thesis, Northumbria University.

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Abstract

Major Histocompatibility Complex (MHC) class I molecules present peptides derived from intracellular proteins for cytotoxic T cell recognition against virally infected cells/ tumor cells and inducing adaptive immunity. MHC-I complexes are comprised of a heavy chain, the light chain beta-2 microglobulin (B2M) and an optimized peptide. Recently, Complex of Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein Cas9 has rapidly become a powerful tool for gene-editing experiments due to its high efficiency, simplicity, and versatility. This thesis proposes two approaches to apply CRISPR/Cas9-mediated knock-in methodology to modify MHC-I complexes: (i) to stably introduce specific peptides on MHC-I molecules through CRISPR-mediated insertion within the B2M locus; (ii) target single amino acid residues of the Ankylosing Spondylitis (AS)-associated HLA-B27 molecule proposed to contribute to its unusual biochemical folding characteristics.

CRISPR/Cas9 was successfully employed to knock in HLA-A2 specific viral peptides, derived from SARS-CoV-2 virus, containing linker sequences at the genomic B2M locus. Immunoblotting revealed that the gene edited B2M locus facilitates the production of peptide linked β2m (PLB) protein with the expected size. Furthermore, flow cytometry analysis demonstrated gene-edited cells presented the PLB molecules together with endogenous peptide-specific HLA-A2 heavy chain at the cell surface.

CRISPR/Cas9 was also harnessed to introduce point mutations into the disease-associated HLA-B*27:05 allele. The absence of Cys67 can potentially reduce the misfolding events of HLA-B27 molecules and still maintain the presence of this heavy chain at the cell surface. Additionally, HLA-B*27:05 have been transformed into HLA-B*27:09, a non-AS-associated subtype, by replacing aspartic acid (Asp)116 with a histidine (His) using CRISPR/Cas9.

This is the first study exploiting gene-editing technology for modification of MHC-I complexes. While stable expression of PLB is a promising approach for development of cellular vaccines, alterations of HLA class I sequences can be considered as a potential therapeutic application for some HLA-associated diseases.

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