Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34⁺CD41⁺CD235ab⁺ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers—CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.

中文翻译：

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通过 CRISPR/Cas9 基因编辑的人类诱导多能干细胞生成缺乏一种或多种血型系统的“设计成红细胞”


尽管输血医学最近取得了进展，但红细胞（RBC）同种免疫对于经产妇女和长期输血患者仍然是一个挑战。有时，诊断实验室依靠难以获得的稀有试剂红细胞来鉴定此类受试者中的不同同种抗体。我们通过使用人类诱导多能干细胞 (hiPSC) 亲本系 (OT1-1) 的 CRISPR/Cas9 基因编辑开发具有定制表型（Rh null、GPB null 和 Kx null/Kell low）的红细胞来解决这个问题。血型系统基因： RHAG 、 GYPB和XK 。将向导RNA克隆到Cas9-嘌呤霉素表达载体中并转染到OT1-1中。进行基因分型以选择嘌呤霉素抗性 hiPSC KO。 CRISPR/Cas9 基因编辑成功产生了三个 KO 系： RHAG KO、 GYPB KO 和XK KO。 OT1-1 细胞系以及三个 KO hiPSC 系分化为 CD34 ⁺ CD41 ⁺ CD235ab ⁺造血祖细胞 (HPC)，随后分化为成红细胞。天然OT1-1成红细胞Rh、MNS、Kell和H血型系统表达呈阳性。 RHAG KO、 GYPB KO和XK KO的分化分别导致Rh null、GPB null和Kx null/Kell低成红细胞的形成。 OT1-1 以及三个 KO 成红细胞的 RBC 标记物 CD71 和 BAND3 仍呈阳性。有红细胞大多处于分化的多色/正色阶段。观察到来自 HPC 的成红细胞增加了约 400 倍。 由 CRISPR/Cas9 基因编辑的 hiPSC 生成的定制成红细胞的可用性应该是对目前用于检测临床重要红细胞同种抗体的工具的有用补充。