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Multiple Knockout of Classical HLA Class II beta-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA
- Source :
- Journal of Immunology, 202(6), 1895-1903. AMER ASSOC IMMUNOLOGISTS
- Publication Year :
- 2019
- Publisher :
- AMER ASSOC IMMUNOLOGISTS, 2019.
-
Abstract
- Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing–based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II–expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.
- Subjects :
- Gene Editing
Genetics
Cas9
HLA-DR beta-Chains
Immunology
Medizin
RNA
Human leukocyte antigen
Biology
Gene Knockout Techniques
03 medical and health sciences
Exon
0302 clinical medicine
Genome editing
CRISPR-Associated Protein 9
Cell Line, Tumor
Humans
Immunology and Allergy
CRISPR
Guide RNA
CRISPR-Cas Systems
Gene
RNA, Guide, Kinetoplastida
030215 immunology
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Journal of Immunology, 202(6), 1895-1903. AMER ASSOC IMMUNOLOGISTS
- Accession number :
- edsair.doi.dedup.....fcd5e7815fa3f2a32542768bd30c7de2