摘要
目的:利用CRISPR/Cas9技术对K562细胞系JAK2基因进行编辑,构建JAK2基因敲除的K562细胞系。方法:使用CRISPR在线设计工具,针对JAK2基因设计sgRNA,构建Cas9-sgRNA共表达质粒。使用第二代慢病毒包装系统包装慢病毒并感染K562细胞,提取细胞基因组DNA,Sanger测序和TA克隆检测基因编辑活性。无限稀释法将编辑阳性的细胞接种于96孔板并扩培得到单克隆细胞株,提取基因组DNA,Sanger测序和TA克隆分析敲除JAK2单克隆细胞的基因型。结果:成功构建靶向敲除JAK2基因的lentiCRISPRv2-sgRNA3-1质粒。优化方案得到低细胞毒性高转染效率的感染K562细胞慢病毒量。CRISPR/Cas9系统成功在JAK2基因sgRNA3-1识别位点发挥基因组编辑活性,获得纯合敲除JAK2基因细胞株K562-JAK2-/-(两个等位分别发生移码突变,预期编码没有功能的JAK2蛋白)。结论:CRIAPR/Cas9系统通过慢病毒感染方式获得JAK2基因纯合敲除的K562细胞株,该细胞模型可用于研究在慢性髓系白血病中JAK2基因的作用,为构建K562敲除其他基因细胞系提供实验依据,为探究造血分化机制的研究奠定实验基础。
Objective:To generate a JAK2 knockout K562 cell line by CRISPR/Cas9 gene editing system.Methods:Using CRISPR online design tool,sgRNA was designed targeted to JAK2 gene and used to construct the co-expression plasmid of Cas9-sgRNA.Lentivirus was packaged by the second-generation lentivirus packaging system and used to transduce K562 cells,the genomic DNA of cell pool was extracted,and the gene editing activity was detected by Sanger sequencing and TA cloning.The candidate edited cells were inoculated into 96-well plate by infinite dilution method.Then the cells were expanded to extract genomic DNA.The JAK2 sequence was identified by Sanger sequencing and TA cloning.Results:LentiCRISPRv2-sgRNA3-1 plasmid containing the gene editing tools for koncking out JAK2 was constructed.The optimal dose of lentivirus with low cytotoxicity and high transduction efficiency was obtained.The JAK2 gene knockout K562 cell line(K562-JAK2-/-)was successfully generated.Conclusion:Lentivirus transduction-based CRISPR/Cas9 system was successfully used to generate JAK2 gene homozygous knockout K562 cell line,providing a cell model to study the importance of JAK2 gene in the field of chronic myeloid leukemia.Besides,the lentivirus transduction-based CRISPR/Cas9 protocol reported here lays a foundation for constructing other gene knockout K562 cell lines to study of hematopoietic differentiation mechanism.
作者
菅璐
黄映辉
梁天亚
王利敏
马洪涛
张婷
李丹阳
王明连
JIAN Lu;HUANG Ying-hui;LIANG Tian-ya;WANG Li-min;MA Hong-tao;ZHANG Ting;LI Dan-yang;WANG Ming-lian(Beijing University of Technology,Beijing 100020,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2019年第7期39-47,共9页
China Biotechnology
基金
北京市自然科学基金面上项目(7182011)资助项目
