利用CRISPR/Cas9系统建立长链非编码RNA SNHG1基因敲除细胞系并研究其功能

Construction of long noncoding RNA SNHG1 knockout cell line using CRISPR/Cas9 system and its function detection

目的利用CRISPR/Cas9介导的同源定向修复机制构建长链非编码RNA(lncRNA)核仁小分子RNA宿主基因1(SNHG1)稳定敲除的A549细胞系,并进行功能研究。方法设计靶向SNHG1基因的小向导RNA(sgRNA),将其克隆到LentiCRISPR v2质粒中,构建Cas9‐sgSNHG1质粒。以A549细胞基因组DNA为模板,PCR扩增5′同源臂(5′Arm)和3′同源臂(3′Arm)序列,以pEGFP‐Blast质粒为模板,PCR扩增pEGFP‐Blast‐polyA序列;利用重叠PCR将3个序列连接为5′Arm‐pEGFP‐Blast‐polyA‐3′Arm,通过Gibson克隆将其连接到pUC18质粒上,构建SNHG1同源重组模板质粒。将构建成功的两个重组质粒共转染A549细胞,用杀稻瘟菌素(Blast)筛选稳定插入pEGFP‐Blast‐polyA的细胞株,挑取单克隆。实时荧光定量PCR(qPCR)检测敲除效率,CCK‐8法和克隆形成实验检测细胞增殖活力及克隆形成能力。结果成功构建Cas9‐sgSNHG1及SNHG1同源重组模板质粒,筛选获得敲除效率较高的A549细胞系,发现敲除SNHG1可抑制A549细胞增殖活力及克隆形成能力。结论SNHG1稳定敲除的A549细胞系构建成功,SNHG1的癌基因功能受到显著抑制。CRISPR/Cas9介导的同源定向修复可有效抑制长链非编码RNA的表达,是长链非编码RNA功能研究的重要工具。

Objective To construct a long noncoding RNA(lncRNA)small nucleolar RNA host gene 1(SNHG1)knockout A549 cell line using the CRISPR/Cas9‐mediated homology‐directed repair mechanism and to detect the biological function of SNHG1.Methods Small guide RNA(sgRNA)targeting the SNHG1 gene was designed and cloned into the LentiCRISPR v2 plasmid,named Cas9‐sgSNHG1 plasmid.The 5′homology arm(5′Arm)and 3′homology arm(3′Arm)sequences were amplified from A549 cell genome DNA while the pEGFP‐Blast‐polyA sequence was amplified from pEGFP‐Blast plasmid.Three sequences were linked as 5′Arm‐pEGFP‐Blast‐polyA‐3′Arm by overlap PCR,and the product was cloned into pUC18 plasmid by Gibson Assembly,named SNHG1 homologous recombinant template plasmid.The two recombinant plasmids were co‐transfected into A549 cells before cell lines stably inserted into pEGFP‐Blast‐polyA were screened with Blasticidin(Blast).Real‐time quantitative fluorescence PCR(qPCR)was used to detect the knockout efficiency of SNHG1,while CCK‐8 and colony formation assay were used to detect cell proliferation and the ability of clone formation.Results Cas9‐sgSNHG1 and SNHG1 homologous recombinant template plasmids were constructed,and A549cell lines with high knockout efficiency were screened.SNHG1 knockout inhibited the proliferation and clone formation of A549 cells.Conclusion SNHG1 knockout A549 cell lines have been constructed,and the function of SNHG1 oncogene is significantly inhibited.CRISPR/Cas9‐mediated homology‐directed repair can effectively inhibit the expression of long noncoding RNA,which is an important tool for studying long noncoding RNA function.