用于全基因组易位测序的Nalm6-Cas9细胞系的构建

Construction of Nalm6-Cas9 Cell Line for Genome-Wide Translocation Sequencing

目的:为进行基于CRISPR-Cas9的全基因组高通量易位测序,利用慢病毒转导构建表达Cas9蛋白的Nalm6-Cas9单克隆细胞系。方法:使用慢病毒载体lentiCas9-Blast、psPAX2和pMD2.G共转染HEK293T细胞包装病毒,病毒感染Nalm6细胞后使用杀稻瘟菌素(Blasticidin)筛选,通过有限稀释法获得多个表达Cas9蛋白的单克隆细胞株。Western blot检测单克隆细胞株中Cas9蛋白的表达水平,细胞计数检测单克隆细胞株的细胞增殖活性。构建lentiCRISPRv2GFP-Δcas9、lentiCRISPRv2GFP-Δcas9-AF4、lentiCRISPRv2GFP-Δcas9-MLL质粒,并分别与pSPAX2、pMD2.G质粒共转染包装病毒,收集病毒感染Nalm6-Cas9单克隆细胞株,使用T载体克隆检测单克隆细胞株的Cas9蛋白的功能。结果:Western blot结果显示,Nalm6-Cas9_1-6单克隆细胞株高水平表达Cas9蛋白;细胞计数分析结果显示,Nalm6-Cas9_1-6单克隆细胞株细胞增殖活性与对照细胞比较无显著差异;T载体克隆检测显示,Nalm6-Cas9_1-6单克隆细胞株Cas9蛋白具有高水平切割活性,对AF4和MLL基因的编辑效率在90%以上。结论:本研究得到了稳定表达高活性Cas9蛋白的Nalm6-Cas9_1-6单克隆细胞株,为基于CRISPR/Cas9全基因组高通量易位测序技术探究治疗相关性白血病中MLL易位连接机制提供依据。

Objective:In order to conduct high-throughput genome-wide translocation sequencing based on CRISPR/Cas9,Nalm6-cas9monoclonal cell line expressing Cas9protein was constructed by lentivirus transduction. Methods:Lentiviral vectors LentiCas9-Blast,pSPAX2,and pMD2. G were used to co-transfect HEK293T cells to obtain recombinant lentivirus. After Nalm6cells were infected with the recombinant lentivirus,the cells were screened by Blasticidin,and multiple monoclonal cell lines expressing Cas9protein were obtained by limited dilution. Western blot was used to detect the expression level of Cas9protein in monoclonal cell lines,and cell count analysis was used to detect the proliferation activity of monoclonal cell lines. LentiCRISPRV2GFP-Δcas9,LentiCRISPRV2GFP-Δcas9-AF4,LentiCRISPRV2GFP-Δcas9-MLL plasmids were constructed,and transfected with pSPAX2and pMD2. G,respectively.T vector cloning was used to detect the function of Cas9protein in Nalm6-Cas9monoclonal cell line infected with virus.Results:Western blot showed that Nalm6-Cas9_1-6monoclonal cell line had high expression of Cas9protein. Cell count analysis showed that high expression of Cas9protein in Nalm6-Cas9 _1-6monoclonal cell line did not affect cell proliferation activity. The Nalm6-Cas9_1-6monoclonal cell line had high cleavage activity,and the editing efficiency of AF4 and MLL genes was more than90% which was determined by T vector cloning. Conclusion:Nalm6-Cas9_1-6monoclonal cell line stably expressing highly active Cas9protein was obtained,which provided a basis for exploring the translocation of MLL in therapy-related leukemias based on CRISPR/Cas9genome-wide high-throughput genome-wide translocation sequencing.