Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
Toronto General Hospital Research Institute, Ajmera Transplant Centre, University Health Network, Toronto, ON, Canada.
Front Immunol. 2023 Oct 5;14:1275413. doi: 10.3389/fimmu.2023.1275413. eCollection 2023.
Innate lymphoid cells (ILCs) are a family of innate lymphocytes with important roles in immune response coordination and maintenance of tissue homeostasis. The ILC family includes group 1 (ILC1s), group 2 (ILC2s) and group 3 (ILC3s) 'helper' ILCs, as well as cytotoxic Natural Killer (NK) cells. Study of helper ILCs in humans presents several challenges, including their low proportions in peripheral blood or needing access to rare samples to study tissue resident ILC populations. In addition, the lack of established protocols harnessing genetic manipulation platforms has limited the ability to explore molecular mechanism regulating human helper ILC biology. CRISPR/Cas9 is an efficient genome editing tool that enables the knockout of genes of interest, and is commonly used to study molecular regulation of many immune cell types. Here, we developed methods to efficiently knockout genes of interest in human ILC2s. We discuss challenges and lessons learned from our CRISPR/Cas9 gene editing optimizations using a nucleofection transfection approach and test a range of conditions and nucleofection settings to obtain a protocol that achieves effective and stable gene knockout while maintaining optimal cell viability. Using IL-4 as a representative target, we compare different ribonucleoprotein configurations, as well as assess effects of length of time in culture and other parameters that impact CRISPR/Cas9 transfection efficiency. Collectively, we detail a CRISPR/Cas9 protocol for efficient genetic knockout to aid in studying molecular mechanism regulating human ILC2s.
先天淋巴细胞 (ILC) 是一类先天淋巴细胞，在免疫反应协调和组织稳态维持中具有重要作用。ILC 家族包括 1 组 (ILC1s)、2 组 (ILC2s) 和 3 组 (ILC3s)“辅助”ILC 以及细胞毒性自然杀伤 (NK) 细胞。研究人类辅助 ILC 存在一些挑战，包括它们在外周血中的比例较低，或者需要访问稀有样本以研究组织驻留 ILC 群体。此外，缺乏利用遗传操作平台的既定方案限制了探索调节人类辅助 ILC 生物学的分子机制的能力。CRISPR/Cas9 是一种高效的基因组编辑工具，可实现感兴趣基因的敲除，常用于研究许多免疫细胞类型的分子调控。在这里，我们开发了在人 ILC2 中有效敲除感兴趣基因的方法。我们讨论了使用核转染转染方法进行 CRISPR/Cas9 基因编辑优化所面临的挑战和经验教训，并测试了一系列条件和核转染设置，以获得一种既能实现有效和稳定的基因敲除，又能保持最佳细胞活力的方案。使用 IL-4 作为代表性靶标，我们比较了不同的核糖核蛋白构型，并评估了培养时间长短和其他影响 CRISPR/Cas9 转染效率的参数的影响。总之，我们详细介绍了一种用于高效遗传敲除的 CRISPR/Cas9 方案，以帮助研究调节人类 ILC2 的分子机制。
