急性髓系白血病是一种复杂多样的疾病，其特征为未分化的髓样前体细胞的克隆扩张。由于造血细胞转染困难，最近已经开发了几种使用CRISPR/Cas9和病毒载体的血液学模型。在本研究中，我们使用融合PCR开发了一种替代策略，以便生成CRISPR构建物，任何配备基本设备的实验室都可以实施。我们通过PCR生成的构建物容易引入难转染的白血病细胞，并通过将MYBL2和IDH2基因添加到HEK293细胞中对其功能进行了双重验证。随后，我们成功地修改了NB4和HL60细胞中明确表达Cas9核酸酶的MYBL2基因，并在IDH2基因中引入R172突变。我们的方法引入突变的效率与核糖核蛋白策略相似，并且未检测到非特异性靶点。总体而言，我们的策略代表着一种有效且直观的方法，可以在不使用病毒传导的情况下将所需的突变引入难转染的白血病细胞中。

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.