Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.
Methods Mol Biol. 2024;2826:79-91. doi: 10.1007/978-1-0716-3950-4_7.
CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.
CRISPR-Cas9 基因组编辑是评估候选基因功能作用的强大工具。体外 CRISPR/Cas9 筛选已被用于快速评估数千个基因在免疫群体分化和功能中的作用。然而，基因的生理相关性通常取决于组织微环境中接收到的信号，例如生长因子、趋化因子、细胞因子和细胞接触依赖性信号的暴露，这些信号在体外环境中可能无法重现。此外，体外方法不足以诱导所有细胞群体的分化，限制了可筛选的细胞类型。这对理解调控生发中心 B 细胞分化的基因构成了重大障碍。在这里，我们描述了一种在激活的 B 细胞中进行体内 Crispr-Cas9 筛选以特异性敲除基因的方法。使用这种方法，我们已经能够揭示病毒感染后生发中心 B 细胞分化的新的转录调控因子。
