Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA.

Journal of immunology (Baltimore, Md. : 1950) 2019 Mar 15

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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. Copyright © 2019 by The American Association of Immunologists, Inc.

Citation

Pietro Crivello, Müberra Ahci, Fabienne Maaßen, Natalie Wossidlo, Esteban Arrieta-Bolaños, Andreas Heinold, Vinzenz Lange, J H Frederik Falkenburg, Peter A Horn, Katharina Fleischhauer, Stefan Heinrichs. Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA. Journal of immunology (Baltimore, Md. : 1950). 2019 Mar 15;202(6):1895-1903