与需要细胞裂解的基于测序的方法不同，光学混合遗传筛选可以研究空间表型，包括细胞形态、蛋白质亚细胞定位、细胞间相互作用和组织组织，以响应靶向 CRISPR 扰动。在这里，我们报告了一种多模态光学混合 CRISPR 筛选方法，我们称之为 CRISPRmap。 CRISPRmap 将原位 CRISPR 引导识别条形码读出与多重免疫荧光和 RNA 检测相结合。通过 DNA 寡核苷酸的组合杂交来检测和读出条形码，提高条形码检测效率。 CRISPRmap能够在传统光学混合筛选难以捉摸的细胞类型和环境中进行原位条形码读出，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体内细胞。我们在乳腺癌细胞系中进行了一项筛选，了解 DNA 损伤修复基因变异对细胞对常用癌症疗法的反应的影响，并且我们表明，光学表型分析可以查明可能的致病性患者衍生突变，这些突变以前被归类为未知临床变异重要性。© 2024。作者。

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.© 2024. The Author(s).