文章：

基于PCR策略将CRISPR/Cas9系统导入造血细胞系

PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines

原文发布日期：25 August 2023

DOI: 10.3390/cancers15174263

类型: Article

开放获取: 是

英文摘要：

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.

摘要翻译： 

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，近期研究利用病毒载体结合CRISPR/Cas9技术开发了多种血液学模型。本研究开发了一种替代策略，通过融合PCR技术构建CRISPR载体，任何配备基础设备的实验室均可实施。我们通过PCR构建的载体能轻松导入难以转染的白血病细胞，并通过在HEK293细胞中导入MYBL2和IDH2基因进行了双重功能验证。随后，我们在持续表达Cas9核酸酶的NB4和HL60细胞中成功修饰了MYBL2基因，并将R172突变引入IDH2基因。该方法的突变导入效率与核糖核蛋白策略相当，且未检测到脱靶事件。总体而言，我们的策略为无需病毒转导即可在难转染白血病细胞中引入目标突变提供了有效且直观的替代方案。

原文链接：

PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines