ThinkCyte Inc., Tokyo 113-8654, Japan.
School of Biomedical Engineering, Faculty of Medicine and Faculty of Applied Science, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Cell Rep Methods. 2024 Mar 25;4(3):100737. doi: 10.1016/j.crmeth.2024.100737.
Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.
基于图像的 pooled CRISPR 筛选的最新进展促进了哺乳动物细胞中多样化基因型-表型关联的映射。然而，基于形态信息的细胞快速富集仍然是一个挑战，限制了在多样化的高内涵细胞表型中进行大规模基因扰动筛选的能力。在这项研究中，我们展示了基于多模态幽灵细胞计数的细胞分选的适用性，包括荧光和无标记的高内涵表型，用于在庞大的细胞群体中进行快速 pooled CRISPR 筛选。我们使用在荧光模式下运行的高内涵细胞分选器，成功地执行了针对影响 RelA 核易位的基因的激酶特异性 CRISPR 筛选。此外，我们使用多参数、无标记模式进行了大规模筛选，以鉴定参与巨噬细胞极化的基因。值得注意的是，无标记平台可以在不进行侵入性染色的情况下富集目标表型，为下游检测保留未受干扰的细胞，并扩展筛选细胞表型的潜力，即使没有合适的标记物。
