美国加州大学圣地亚哥分校Gene W. Yeo研究团队取得一项新突破。他们利用微筏阵列成像结合CRISPR筛选库揭示了应力颗粒调节因子。相关论文于2020年5月11日在线发表于《自然—方法学》。

研究人员将CRISPR–Cas9筛选库与微筏阵列技术和高内涵成像相结合，以筛选基于图像的表型（CRaft-ID；基于CRISPR的微筏阵列，然后进行指导RNA（gRNA）鉴定）。

通过分离包含具有单个gRNA的基因克隆微筏，研究人员确定了影响应激颗粒形成的RNA结合蛋白（RBP），其是应激过程中形成的点状蛋白质-RNA组装体。为了自动进行靶点识别，研究人员开发了一种机器学习模型，其依据已有的核形态学来去除不健康的细胞或成像伪像。

在此过程中，研究人员发现并验证了以前无法表征的RBP，这些RBP可以调节应激颗粒的丰度，从而突出了该方法可基于图像进行CRISPR筛选的便利性 。

研究人员表示，使用CRISPR库的遗传筛选方法具有可扩展性和廉价的特点，但其受限于标准读数，包括生存、增殖和可分类标记。但是，许多生物学上相关的细胞状态都涉及细胞和亚细胞的变化，这些变化只能通过显微镜才能观察到，并且目前无法用合并方法进行筛选。

附：英文原文

Title: Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors

Author: Emily C. Wheeler, Anthony Q. Vu, Jaclyn M. Einstein, Matthew DiSalvo, Noorsher Ahmed, Eric L. Van Nostrand, Alexander A. Shishkin, Wenhao Jin, Nancy L. Allbritton, Gene W. Yeo

Issue&Volume: 2020-05-11

Abstract: Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR–Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein–RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.

DOI: 10.1038/s41592-020-0826-8

Source: https://www.nature.com/articles/s41592-020-0826-8