DataSheet_1_Efficient and stable CRISPR/Cas9-mediated genome-editing of human type 2 innate lymphoid cells.pdf

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.

天然淋巴细胞（ILCs）是一类在免疫反应协调和组织稳态维持中发挥关键作用的固有淋巴细胞。ILC家族包括第1组（ILC1s）、第2组（ILC2s）和第3组（ILC3s）的辅助性ILC，以及细胞毒性自然杀伤（NK）细胞。人类辅助性ILC的研究面临诸多挑战，包括其在外周血中的比例较低或需获取罕见样本以研究组织定居的ILC群体。此外，缺乏利用基因操作平台建立的成熟协议，限制了探索调节人类辅助性ILC生物学分子机制的能力。CRISPR/Cas9是一种高效的基因组编辑工具，能够实现对目标基因的敲除，并常用于研究多种免疫细胞类型的分子调节。在本研究中，我们开发了针对人类ILC2s高效敲除目标基因的方法。我们讨论了通过核转染方法进行CRISPR/Cas9基因编辑优化过程中遇到挑战和汲取的教训，并测试了一系列条件和核转染设置，以获得一个既能实现有效且稳定的基因敲除又能保持最佳细胞存活率的方案。以IL-4作为代表靶标，我们比较了不同的核糖核蛋白配置，并评估了培养时间长度以及其他影响CRISPR/Cas9转染效率的参数。总之，我们详细描述了一种CRISPR/Cas9方案，以实现高效的基因敲除，从而有助于研究调节人类ILC2s分子机制的分子机制。