利用CRISPR/Cas9技术构建敲除MEIS2基因的HEK293T人胚肾细胞系  

Construction of MEIS2 Knockout HEK293T Cell Line by CRISPR/Cas9 Technology

作　　者：卢利莎[1] 白杨[2] 刘鑫[2] 王洪涛[2] 高洁[2] 杨亦青[2,3] 苏培[2] 刘翠翠[2] 王钰[2] 张磊升[2] 熊涛[1] 周家喜[2] 

机构地区：[1]长江大学生命科学学院,荆州434025 [2]中国医学科学院北京协和医学院血液病医院(血液学研究所),实验血液学国家重点实验室,天津300020 [3]河北北方学院医学检验学院,张家口075000

出　　处：《中国细胞生物学学报》2015年第4期535-541,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31171431;31301206)资助的课题~~

摘　　要：CRISPR/Cas9系统作为一种新型的基因组编辑技术,利用人工设计的向导RNA(singleguide RNA,sg RNA)介导外源表达的Cas9蛋白与基因组靶点特异性结合以实现对基因组DNA的特异性切割,切割后的基因组DNA通过非同源末端连接或同源重组的方式进行修复,从而实现基因的敲除、敲入等。MEIS2属于一类高度保守的同源盒转录因子MEIS家族,研究发现,MEIS2广泛参与胚胎的早期发育及肿瘤的发生发展,但其发挥作用的机制目前还不是很清楚。该研究针对MEIS2基因作用的功能域,设计两个靶向MEIS2基因Exon3和Exon8的sg RNA,通过SURVEYOR分析及Western blot检测,确认了所设计sg RNA的有效性。进一步通过细胞分选及Western blot检测筛选出稳定敲除MEIS2基因的HEK293T细胞株。最后,通过序列测定确认MEIS2发生了移码突变。综上所述,该研究利用CRISPR/Cas9技术成功建立了完全敲除MEIS2的HEK293T细胞株,为研究MEIS2的功能和作用机制提供了有效工具。CRISPR/Cas9 system is the newest gene-editing technology through constitutive expression of nucleases Cas9, which binds to specific site in the genome mediated by single-guide RNA and induces doublestrand breaks（DSBs） at desired genomic loci. DSBs induced by these site-specific nucleases can be repaired by error-prone nonhomologous end joining（NHEJ） or homology directed repair（HDR）, to generate gene-specific knockout or knockin cells. MEIS2 belongs to MEIS transcription factor family with conservative homeodomains. Studies have suggested that MEIS2 is a potent regulator of development and tumorigenesis, but the mechanisms are still unclear. In this study, we designed two single-guide RNAs targeting the Exon3 and Exon8 of MEIS2 gene, which encode its two homeodomains. We measured their gene-targeting efficiency by SURVEYOR assay and Western blot analysis. Through dilution plating, we isolated a monoclonal MEIS2 knockout cell line. Sequence analysis indicated that it contained freamshift mutations in both MEIS2 alleles. In summary, we have successfully employed CRISPR/Cas9 system to construct a MEIS2 knockout cell line, and it will be a powerful tool for studying the functions and related mechanisms of MEIS2.

关 键 词：MEIS2 CRISPR/Cas9系统 基因敲除 

分 类 号：Q78[生物学—分子生物学]