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• W2889161676 abstract "The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. Here we investigate a questionable (Q) allele HLA-B∗38:68Q, which carries a 9-nucleotide (nt) deletion at codon 230–232 on exon 4 (Fig. 1A), using CRISPR/Cas9 gene editing to study the effect of this mutation on HLA-B∗38:68Q expression. The CRISPR/Cas9, targeting gRNA and a non-PAM (Protospacer Adjacent Motif) ssDNA oligo were co-transfected to EBV-B cell line TEM665 (Fig. 1B) by Neon (Thermo Fisher Scientific) to generate homozygous deletions at codon 230–232 of HLA-B∗38:01:01. After single cell seeding and expanding, Sanger sequencing and next generation sequencing were performed to validate gene editing in single cell derived clones. Cell surface expression was determined by flow cytometric analysis using a FITC–conjugated Bw4 IgG antibody (One Lambda). The gene editing of HLA-B∗38:01:01 homozygous EBV-B cell line by CRISPR/Cas9 resulted in 25 single cell derived clones. The data showed that 21/25 (84%) clones were successfully edited. Among the edited alleles, 5 alleles were through homology directed repair (HDR) pathway and 36 alleles were through non homologous end joining (NHEJ) pathway (Fig. 1C). We acquired two precise edited clones: one was HLA-B∗38:68Q/B∗38:01:01 heterozygous and the other one was HLA-B∗38:68Q homozygous (Fig. 1D). Based on the flow cytometric analysis (Fig. 1E), the expression of HLA-B∗38:01:01 homozygous was 2.2-fold higher than HLA-B∗38:68Q/B∗38:01:01 heterozygous cells, and the HLA-B∗38:68Q/B∗38:01:01 heterozygous cells was over 2.0-fold higher than HLA-B∗38:68Q homozygous. We demonstrated that HLA-B∗38:68Q is a low-expressed allele using CRISPR/Cas9 gene editing. CRISPR/Cas9 can induce precise gene editing in HLA genes. It provides a useful tool to study the functions of insertion, deletion and intron sequences of HLA genes.Download : Download high-res image (940KB)Download : Download full-size image" @default.
• W2889161676 created "2018-09-07" @default.
• W2889161676 creator A5031963052 @default.
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• W2889161676 date "2018-10-01" @default.
• W2889161676 modified "2023-09-25" @default.
• W2889161676 title "OR19 Integrate crispr/CAS9 for precise protein expression of HLA-B∗38:68Q" @default.
• W2889161676 doi "https://doi.org/10.1016/j.humimm.2018.07.024" @default.
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