Department of Pathology.
Bass Center for Childhood Cancer and Blood Disorders, Department of Pediatrics.
Blood Adv. 2019 Oct 8;3(19):2825-2835. doi: 10.1182/bloodadvances.2019000450.
Chromosomal rearrangements involving the mixed lineage leukemia () gene, also known as , are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that -rearranged () mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.
涉及混合谱系白血病（）基因的染色体重排，也称为，在人类白血病中经常观察到，通常与预后不良有关。为了模拟这些白血病，我们应用成簇规律间隔短回文重复（CRISPR）/Cas9 基因编辑在从脐带血中纯化的人类造血干祖细胞中诱导染色体重排。包含化学修饰的合成单指导 RNA 和纯化的 Cas9 蛋白的核糖核蛋白复合物的电穿孔诱导 9 号和 11 号染色体之间的易位 [t（9;11）]，效率>1%。基因编辑细胞移植到免疫缺陷小鼠中迅速诱导不同谱系的急性白血病，并且通常具有多克隆起源，这取决于移植前体外培养的时间。断点连接序列作为生物标志物，用于监测体外和体内培养中的克隆选择和进展。通过质谱流式细胞术（CyTOF）进行的高维细胞表面和细胞内蛋白质分析表明，基因编辑的白血病再现了人类患者中观察到的疾病特异性蛋白质表达，并表明 -重排（）混合表型急性白血病（MPAL）与急性髓细胞性白血病（AML）比急性淋巴细胞性白血病（ALL）更相似。因此，使用 CRISPR/Cas9 在原代人类血液干细胞中高效产生染色体易位可靠地模拟人类急性白血病，并为白血病生物学和治疗学的基础和转化研究提供了实验平台。
