Single-cell CRISPR screening characterizes transcriptional deregulation in T-cell acute lymphoblastic leukemia. Single-cell CRISPR screening characterizes transcriptional deregulation in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia caused by accumulation of genetic alterations in T-cell progenitors. For many genes it remains unknown how loss-of-function mutations contribute to disease development. Single-cell CRISPR screening in ex vivo pro-T cells allowed us to study the transcriptomic impact of such alterations. A first CRISPR screen targeted 17 well-studied regulators and defined the core transcriptional signatures, such as NOTCH1, MYC, STAT5 and E2F. Additionally, Spi1 was identified as an essential gene associated with the MYC regulon. A second screen targeted 42 poorly characterized genes and identified gene clusters with E2F/MYC and STAT/NOTCH1 signatures having opposing roles. Bcl11b inactivation conferred the strongest growth advantage and was associated with JAK/STAT upregulation, corresponding with publicly available T-ALL patient data. Bcl11b inactivation cooperated with mutant JAK3 in transforming pro-T cells to cytokine-independent growth. With this data, we characterized tumor suppressors and oncogenes in T-ALL, thereby providing insight in the mechanisms of leukemia development. Overall design: ex-vivo murine ProT cells were transduced with a CRISPR library and analysed using the 10x genomics feature-barcoding technology (capture of both mRNA and CRISPR guides)

T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）是一类侵袭性白血病，由T细胞祖细胞内遗传变异累积所引发。目前，多数基因的功能丧失性突变如何参与疾病发生发展的机制仍未明确。
我们通过对离体原T细胞（ex vivo pro-T cells）开展单细胞CRISPR筛选（single-cell CRISPR screening），得以解析这类遗传变异的转录组学效应。首轮CRISPR筛选靶向了17个已被充分研究的调控因子，明确了核心转录特征，涵盖NOTCH1、MYC、STAT5及E2F等通路。此外，本研究还鉴定出Spi1为与MYC调控子（MYC regulon）相关的必需基因。
第二轮CRISPR筛选靶向了42个功能未被充分表征的基因，鉴定出带有E2F/MYC与STAT/NOTCH1特征的基因簇，二者发挥相反的调控作用。Bcl11b失活可赋予细胞最强的生长优势，且与JAK/STAT通路上调密切相关，这一结果与公开的T-ALL患者数据集相契合。进一步研究发现，Bcl11b失活可与突变型JAK3协同，将原T细胞转化为不依赖细胞因子生长的细胞。
基于本数据集，我们系统解析了T-ALL中的抑癌基因与致癌基因，为白血病发生发展的机制研究提供了重要见解。
总体设计：将CRISPR文库转导至小鼠离体原T细胞中，随后采用10x Genomics特征条形码技术（可同时捕获mRNA与CRISPR向导RNA）开展分析。