Jiacheng Gu, Abhishek Iyer, Ben Wesley, Angelo Taglialatela, Giuseppe Leuzzi, Sho Hangai, Aubrianna Decker, Ruoyu Gu, Naomi Klickstein, Yuanlong Shuai, Kristina Jankovic, Lucy Parker-Burns, Yinuo Jin, Jia Yi Zhang, Justin Hong, Xiang Niu, Jonathon A. Costa, Mikael G. Pezet, Jacqueline Chou, Hans-Willem Snoeck, Dan A. Landau, Elham Azizi, Edmond M. Chan, Alberto Ciccia, Jellert T. Gaublomme
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引用次数: 0
摘要
基于测序的方法需要裂解细胞,与之不同的是,光学集合基因筛选可以研究细胞形态、蛋白质亚细胞定位、细胞-细胞相互作用和组织组织等空间表型对靶向 CRISPR 干扰的响应。在这里,我们报告了一种多模式光学集合CRISPR筛选方法,我们称之为CRISPRmap。CRISPRmap 将原位 CRISPR 引导识别条形码读取与多重免疫荧光和 RNA 检测相结合。条形码通过 DNA 寡聚物的组合杂交进行检测和读出,从而提高了条形码的检测效率。CRISPRmap 能够在细胞类型和环境中进行原位条形码读取,而传统的光学集合筛选则难以实现,包括培养的原代细胞、胚胎干细胞、诱导多能干细胞、衍生神经元和组织环境中的活体细胞。我们在乳腺癌细胞系中筛选了 DNA 损伤修复基因变异对细胞对常用癌症疗法反应的影响,结果表明,光学表型能准确定位以前被归类为临床意义未知变异的可能致病的患者来源变异。
Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap
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.
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