美国哥伦比亚大学Jellert T. Gaublomme团队使用CRISPRmap将多模态表型映射到细胞和组织中的扰动。这一研究成果于2024年10月7日在线发表在国际学术期刊《自然—生物技术》上。

研究人员报告了一种多模态光学混合CRISPR筛选方法，称为CRISPRmap。CRISPRmap结合了原位CRISPR引导识别条形码的读取、多个免疫荧光标记和RNA检测。通过DNA寡核苷酸的组合杂交检测和读取条形码，从而提高了条形码检测的效率。CRISPRmap能够在以前难以通过传统光学混合筛选检测的细胞类型和背景中实现原位条形码读取，包括培养的原代细胞、胚胎干细胞、诱导多能干细胞、衍生神经元以及组织背景中的体内细胞。

研究人员在一个乳腺癌细胞系中进行了筛选，揭示了DNA损伤修复基因变体对常用癌症治疗的细胞反应的影响。并且研究人员发现，光学表型能够明确可能致病的患者来源突变，这些突变以前被分类为临床意义不明的变体。

据介绍，与需要细胞裂解的测序方法不同，光学混合遗传筛选能够调查空间表型，包括细胞形态、蛋白质亚细胞定位、细胞间相互作用和组织结构，响应于靶向CRISPR扰动。

附：英文原文

Title: Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap

Author: Gu, Jiacheng, Iyer, Abhishek, Wesley, Ben, Taglialatela, Angelo, Leuzzi, Giuseppe, Hangai, Sho, Decker, Aubrianna, Gu, Ruoyu, Klickstein, Naomi, Shuai, Yuanlong, Jankovic, Kristina, Parker-Burns, Lucy, Jin, Yinuo, Zhang, Jia Yi, Hong, Justin, Niu, Xiang, Costa, Jonathon A., Pezet, Mikael G., Chou, Jacqueline, Snoeck, Hans-Willem, Landau, Dan A., Azizi, Elham, Chan, Edmond M., Ciccia, Alberto, Gaublomme, Jellert T.

Issue&Volume: 2024-10-07

Abstract: Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell–cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.

DOI: 10.1038/s41587-024-02386-x

Source: https://www.nature.com/articles/s41587-024-02386-x