Key result
CRISPR/Cas9 genome editing using a single guide RNA achieved simultaneous knockout of HLA-DRB, -DQB1, and -DPB1 with 99.5% penetrance in human B lymphoblastoid cell lines.
A single guide RNA CRISPR/Cas9 approach successfully achieved comprehensive knockout of classical HLA class II beta-chains in human B lymphoblastoid cell lines, enabling mechanistic studies of individual HLA variants.
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Enables HLA-II editing in diverse B-cell lines; leaves open clinical translation pending in vivo and safety data.
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.
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Crivello et al. (2019) studied this question. CRISPR/Cas9 genome editing targeting HLA-DRB, -DQB1, and -DPB1 through a single guide RNA was evaluated on Abrogation of HLA-II surface expression and specific genomic insertion/deletion mutations. CRISPR/Cas9 genome editing using a single guide RNA achieved simultaneous knockout of HLA-DRB, -DQB1, and -DPB1 with 99.5% penetrance in human B lymphoblastoid cell lines.
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