Datlinger Paul, Rendeiro André F, Schmidl Christian, Krausgruber Thomas, Traxler Peter, Klughammer Johanna, Schuster Linda C, Kuchler Amelie, Alpar Donat, Bock Christoph
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
Nat Methods. 2017 Mar;14(3):297-301. doi: 10.1038/nmeth.4177. Epub 2017 Jan 18.
CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.
基于CRISPR的基因筛选正在加速生物学发现，但目前的方法存在固有局限性。广泛使用的混合筛选仅限于简单的读数，包括细胞增殖和可分选的标记蛋白。阵列筛选允许进行全面的分子读数，如转录组分析，但通量要低得多。在这里，我们将混合CRISPR筛选与单细胞RNA测序结合成一个广泛适用的工作流程，直接将导向RNA表达与数千个单个细胞中的转录组反应联系起来。我们的CRISPR液滴测序（CROP-seq）方法能够以单细胞转录组分辨率进行混合CRISPR筛选，这将有助于对复杂调控机制和异质细胞群体进行高通量功能剖析。
