T 细胞急性淋巴细胞白血病 (T-ALL) 是一种侵袭性白血病，由 T 细胞祖细胞中多种遗传改变的积累引起。然而，对于许多基因来说，它们的突变如何导致疾病的发展仍然未知。因此，我们在原代 T 细胞离体中进行了两次单细胞 CRISPR 筛选，以研究 T-ALL 中功能丧失改变的转录影响，并将其与对细胞适应性的影响联系起来。各种扰动根据其对 E2F/MYC 或 STAT/NOTCH 特征的影响进行聚类，这些特征在驱动 T 细胞增殖中发挥着决定性作用。许多扰动会对 STAT 和 NOTCH 特征产生积极影响，并预计在 T-ALL 中表现为单倍体不足的肿瘤抑制因子。此外，Spi1 被确定为前 T 细胞存活的必需基因，与 MYC 特征的失调和表观遗传后果相关。相比之下，Bcl11b 在未成熟 T 淋巴细胞中被鉴定为强抑癌基因，与 NF-kB 和 JAK/STAT 信号传导失调相关。我们发现 T-ALL 患者中 BCL11B 表达水平与 JAK/STAT 通路突变之间存在相关性，并证明了亲 T 细胞中 Bcl11b 失活与 JAK3 过度激活之间的致癌协同作用。总而言之，这些在原 T 细胞中进行的单细胞 CRISPR 筛选为 T-ALL 基因改变引起的转录失调机制提供了基本见解。

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of leukemia caused by accumulation of multiple genetic alterations in T-cell progenitors. However, for many genes it remains unknown how their mutations contribute to disease development. Therefore, we performed two single-cell CRISPR screens in primary pro-T cells ex vivo to study the transcriptional impact of loss-of-function alterations in T-ALL and correlate this with effects on cell fitness. The various perturbations were clustered based on their effects on E2F/MYC or STAT/NOTCH signatures, which play a defining role in driving T-cell proliferation. Many of the perturbations resulted in positive effects on the STAT and NOTCH signatures and were predicted to behave as haploinsufficient tumor suppressors in T-ALL. Additionally, Spi1 was identified as an essential gene for pro-T cell survival, associated with deregulation of the MYC signature and epigenetic consequences. In contrast, Bcl11b was identified a strong tumor suppressor gene in immature T lymphocytes, associated with deregulation of NF-kB and JAK/STAT signaling. We found a correlation between BCL11B expression level and JAK/STAT pathway mutations in T-ALL patients and demonstrated oncogenic cooperation between Bcl11b inactivation and JAK3 hyperactivation in pro-T cells. Altogether, these single-cell CRISPR screens in pro-T cells provide fundamental insights in the mechanisms of transcriptional deregulation caused by genetic alterations in T-ALL.