使用单细胞读数的遗传扰动筛选使得基因功能和调控网络的丰富表型成为可能。这些方法在体内一直具有挑战性，特别是在癌症等成人疾病模型中，其中包括恶性细胞和微环境细胞的混合物。胶质母细胞瘤 (GBM) 是一种致命的癌症，缺乏系统地检测体内基因功能和治疗靶点的方法，特别是与放射治疗等标准护理治疗相结合的方法。在这里，我们迭代开发了一种多重体内扰动序列 CRISPRi用于癌症和肿瘤微环境细胞中单细胞遗传筛选的平台，利用颅内对流增强 sgRNA 文库向 GBM 小鼠模型的递送。我们的平台能够有效沉默体内生长和肿瘤维持的驱动因素以及使 GBM 对放射治疗敏感的基因。我们发现放疗以体内依赖性方式重新连接对遗传扰动的转录反应，揭示了 GBM 治疗敏感性或耐药性的异质模式。此外，我们证明了靶向在肿瘤微环境中发挥作用的基因，能够在体内 CRISPRi 扰动后改变免疫细胞和基质细胞之间的配体-受体相互作用，从而影响肿瘤细胞的吞噬作用。总之，我们证明了多重扰动-seq 的实用性用于在治疗干预的背景下对成人癌症和正常组织生物学进行体内单细胞解剖，这是一个具有广泛应用潜力的平台。© 2024。作者。

Genetic perturbation screens with single-cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking.Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single-cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery of sgRNA libraries into mouse models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo-dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune and stromal cells following in vivo CRISPRi perturbations that can affect tumor cell phagocytosis.In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single-cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.© 2024. The Author(s).