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Fig. 1 | Egyptian Journal of Medical Human Genetics

Fig. 1

From: HLA-A gene knockout using CRISPR/Cas9 system toward overcoming transplantation concerns

Fig. 1

Schematic summary of the process of establishing HLA-A deficient HEK293T cells using dual gRNA CRISPR/Cas9-mediated gene KO. To obtain a large deletion within the HLA-A gene, we co-transfected the cells with two plasmids containing the GFP gene and two different gRNAs. The green GFP+ cells were sorted by FACS after 48 h and then cultured to reach the confluency of 70–90%. After that, a suspension of cells with a defined concentration was provided to have a single cell per well in a 96-well plate. After 7–10 days, the DNAs of single-cell clones were extracted, and the PCR reaction and sequencing results revealed and confirmed the clones with mutations, respectively. Finally, the expression level of mutated clones was analyzed using real-time PCR

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