摘要
【目的】应用CRISPR/Cas9方法,构建特异性靶向长链非编码RNA DANCR基因两端的双靶标慢病毒载体,稳定敲除间充质干细胞(MSC)中的DANCR基因,为研究DANCR的生物学功能奠定基础。【方法】首先设计靶向DANCR的5’和3’端的sgRNA(single-guide RNA),转染293FT细胞,提取293FT细胞基因组DNA,验证靶向序列的有效性。然后通过gateway和酶切连接的方法,将分别靶向5’和3’端的两条有效sgRNA序列连接至同一慢病毒CRISPR载体。最终使用293FT进行慢病毒包装,收获病毒感染MSC,检测MSC中DANCR敲除效率。【结果】在证明靶向DANCR基因两端的sgRNA序列均单独有效的基础上,将靶向5’和3’端的sgRNA进行组合,成功构建靶向DANCR的双靶标慢病毒载体。将载体进行慢病毒包装后感染MSC,成功获得DANCR敲除的MSC。【结论】应用CRISPR方法成功构建敲除DANCR的慢病毒载体,能高效稳定地敲除MSC中的DANCR基因。
【Objective】Using the CRISPR/Cas9(CRISPR/crispr-associated(Cas)9 method,a dual-target lentiviral vector containing single-guide RNAs(sgRNAs)targeting both the 5’and 3’ends of the anti-differentiation noncoding RNA(DANCR)gene was constructed.Stable knockout of DANCR gene in mesenchymal stem cells(MSC)would be help.ful for the future study of the biological function of DANCR.【Methods】Designed sgRNAs targeting either the 5’or 3’end of DANCR and cloned into two CRISPR vectors.The vector was transfected into 293FT cells,and the genomic DNA of 293FT cells was extracted to verify the efficiency of individual sequence.Two functional sgRNAs targeting either the 5’or 3’end were incorporated into a same lentiviral CRISPR vector through gateway and enzymatic ligation.293FT was used for lentiviral packaging,after which the virus was harvested to infect MSC,and the knockout efficiency of DANCR in MSC was detected.【Results】All four sgRNA sequences targeting DANCR successfully guided Cas9 to cleave the gene.sgRNAs targeting either the 5’and 3’end were combined to establish a dual-target lentiviral vector for stable knockout of DANCR.The vector was packaged into lentivirus and infected MSC.Finally,we successfully obtained mesenchymal stem cell lines with DANCR gene knockout.【Conclusions】Using the CRISPR method,a dual-target lentiviral vector can efficiently and stably knock out DANCR gene in MSC.
作者
彭丹
钟小敏
PENG Dan;ZHONG Xiao-min(Center for Stem Cell Biology and Tissue Engineering,Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou 510080,China)
出处
《中山大学学报（医学版）》
CAS
CSCD
北大核心
2019年第1期14-22,共9页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家重点研发计划项目(2017YFA0105501)
广东省科技计划项目(2015A020212019)
