A novel CRISPR activation mouse enables modelling of aggressive lymphoma and interrogation of venetoclax resistance. A novel CRISPR activation mouse enables modelling of aggressive lymphoma and interrogation of venetoclax resistance

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI/+ 38 ) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI 39 primary lymphocytes, we induced B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivated pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ 42 haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly developed lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas were highly sensitive to the BCL-2 inhibitor venetoclax. We performed genome-wide activation screens in these lymphoma cells and found a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the power of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies. Overall design: Comparative gene expression profiling analysis of RNA-seq data for Mouse Embryonic Fibroblast (MEF) cell lines.

CRISPR技术已助力小鼠癌症建模研究的发展，但CRISPR激活（CRISPR activation, CRISPRa）方法尚未在该领域得到应用。我们构建了一种可在体内及体外诱导基因表达的CRISPRa小鼠（dCas9a-SAMKI/+ 38）。利用dCas9a-SAMKI 39原代淋巴细胞，我们可在T细胞中诱导B细胞限制性基因的表达，反之亦然，这彰显了本系统的应用效能。侵袭性双重打击淋巴瘤的现有模型较为匮乏。为此，我们在Eµ-MycT/+;dCas9a-SAMKI/+ 42造血干祖细胞中对促存活基因BCL-2进行了转录激活。移植了上述细胞的小鼠快速出现了高表达BCL-2与MYC的淋巴瘤。与标准Eµ-Myc淋巴瘤不同，表达BCL-2的淋巴瘤对BCL-2抑制剂维奈克拉（venetoclax）高度敏感。我们在这些淋巴瘤细胞中开展了全基因组激活筛选，发现BCL-2家族蛋白A1在维奈克拉耐药性中发挥主导作用。本研究证实了我们的CRISPRa模型在疾病模拟及解析靶向治疗耐药机制方面的重要价值。整体实验设计：对小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblast, MEF）细胞系的RNA测序数据进行比较基因表达谱分析。