利用 RNA 电穿孔一步法 CRISPR-Cas9 介导的人 T 细胞内源性 TCRαβ 基因敲除。
One-step CRISPR-Cas9-mediated knockout of native TCRαβ genes in human T cells using RNA electroporation.
机构信息
Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610 Wilrijk, Belgium.
出版信息
STAR Protoc. 2023 Mar 17;4(1):102112. doi: 10.1016/j.xpro.2023.102112. Epub 2023 Feb 10.
To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRβ chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8 T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRβ constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.
为了避免天然和引入的 T 细胞受体 (TCR) 之间的错配，并防止同种异体 T 细胞治疗中的移植物抗宿主病，通过 CRISPR-Cas9 敲除天然 TCR 的 TCRα 和 TCRβ 链。我们展示了 CD8 T 细胞的分离和激活，然后用体外转录的 eSpCas9(1.1)-P2A-EGFP mRNA 和针对 TCRα 和 TCRβ 恒定区的单指导 RNA 对 T 细胞进行电穿孔。然后，我们描述了一种流式细胞术分析来确定 TCR 敲除效率。
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