RNA-seq data from NSCs and tumorspheres generated using the RCAS/TVA-CRISPR/Cas9 system

It has been gradually established that the vast majority of human tumors are extraordinarily heterogeneous at a genetic level. To accurately recapitulate this complexity, it is now evident that in vivo animal models of cancers will require to recreate not just a handful of simple genetic alterations, but possibly dozens and increasingly intricate. Here, we have combined the RCAS/TVA system with the CRISPR/Cas9 genome editing tools for precise modelling of human tumors. We show that somatic deletion in neural stem cells (NSCs) of a variety of known tumor suppressor genes (Trp53, Cdkn2a and Pten), in combination with the expression of an oncogene driver, leads to high-grade glioma formation. Moreover, by simultaneous delivery of pairs of guide RNAs (gRNAs) we generated different gene fusions, either by chromosomal deletion (Bcan-Ntrk1) or by chromosomal translocation (Myb-Qk), and we show that they have transforming potential in vivo and in vitro. Overall design: Bcan-Ntrk1 tumorspheres, Myb-Qk gRNA transduced NSCs, Ctrl gRNA transduced NSCs, Cdkn2a gRNA PDGFB tumorspheres, Pten gRNA PDGFB tumorspheres, and Trp53 gRNA PDGFB tumorspheres. 2 replicates each.越来越多的研究证实，绝大多数人类肿瘤在遗传层面展现出极高的异质性。为精准重现肿瘤的这一复杂特性，现有研究表明，癌症的体内动物模型不仅需要复刻少量简单的遗传改变，更需要构建数十种乃至愈发复杂的遗传变异。本研究将RCAS/TVA系统与CRISPR/Cas9基因组编辑工具相结合，用于人类肿瘤的精准建模。我们发现，在神经干细胞（NSCs）中对多种已知肿瘤抑制基因（Trp53、Cdkn2a与Pten）进行体细胞缺失，并结合致癌驱动基因的表达，可诱导高级别胶质瘤的形成。此外，通过同时递送向导RNA（gRNAs）对组合，我们分别通过染色体缺失（Bcan-Ntrk1）与染色体易位（Myb-Qk）构建了不同的基因融合产物，并证实其在体内与体外均具有转化潜能。整体实验设计：Bcan-Ntrk1肿瘤球、Myb-Qk向导RNA转导的神经干细胞、对照向导RNA（Ctrl gRNA）转导的神经干细胞、Cdkn2a向导RNA联合PDGFB肿瘤球、Pten向导RNA联合PDGFB肿瘤球，以及Trp53向导RNA联合PDGFB肿瘤球，每组设置2个生物学重复。