Proietti Ludovica, Manhart Gabriele, Heyes Elizabeth, Troester Selina, Grebien Florian
Institute for Medical Biochemistry, University of Veterinary Medicine, Vienna, Austria.
Bio Protoc. 2022 Dec 20;12(24). doi: 10.21769/BioProtoc.4577.
CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50-300 gene knockouts on various cellular parameters within eight weeks. Leukemia (2021), DOI: 10.1038/s41375-021-01169-6 Graphical abstract.
CRISPR/Cas9筛选彻底改变了生物医学研究中的功能基因组学，是一种广泛用于识别癌细胞中基因依赖性的方法。在这里，我们展示了一种高效且通用的方案，用于将引导RNA（gRNA）克隆到慢病毒载体中，生产慢病毒上清液，并以96孔板形式转导靶细胞。为了评估基因敲除对细胞适应性的影响，我们描述了一种基于竞争的细胞增殖测定法，该方法使用流式细胞术，能够以快速且可重复的方式同时筛选多个基因。这种读数可以扩展到基于流式测量可获取的任何参数，例如蛋白质表达和稳定性、分化、细胞死亡等。总之，该方案能够在八周内从功能上评估一组50 - 300个基因敲除对各种细胞参数的影响。《白血病》（2021年），DOI: 10.1038/s41375 - 021 - 01169 - 6 图形摘要。
