González-Romero Elisa, Martínez-Valiente Cristina, García-García Gema, Rosal-Vela Antonio, Millán José María, Sanz Miguel Ángel, Sanz Guillermo, Liquori Alessandro, Cervera José Vicente, Vázquez-Manrique Rafael P
Hematology Research Group, Instituto de Investigación Sanitaria La Fe, 46026 Valencia, Spain.
CIBERONC, 28029 Madrid, Spain.
Cancers (Basel). 2023 Aug 25;15(17):4263. doi: 10.3390/cancers15174263.
Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.
急性髓系白血病是一种复杂的异质性疾病，其特征是未分化髓系前体细胞的克隆性扩增。由于血细胞转染困难，最近利用病毒载体通过CRISPR/Cas9开发了几种血液学模型。在本研究中，我们开发了一种替代策略，通过融合PCR生成CRISPR构建体，任何配备基本设备的实验室都可以实施。我们通过PCR生成的构建体很容易导入难以转染的白血病细胞，并通过在HEK293细胞中添加MYBL2和IDH2基因对其功能进行了双重验证。然后，我们成功地修饰了NB4和HL60细胞中的MYBL2基因，并将R172突变引入到组成型表达Cas9核酸酶的IDH2基因中。我们方法的突变引入效率与核糖核蛋白策略相似，未检测到脱靶事件。总体而言，我们的策略是一种有效且直观的替代方法，可在不进行病毒转导的情况下将所需突变引入难以转染的白血病细胞。
