CRISPR/dCas9-KRAB系统沉默猪LncRNA-NEAT1基因  

Silencing of pig LncRNA-NEAT1 gene by CRISPR/dCas9-KRAB system

作　　者：赵为民[1,2,3] 戴超辉 陈哲 涂枫[1,2,3] 李辉 付言峰[1,2,3] 李碧侠 任守文[1,2,3] 程金花 ZHAO Wei-min;DAI Chao-hui;CHEN Zhe;TU Feng;LI Hui;FU Yan-feng;LI Bi-xia;REN Shou-wen;CHENG Jin-hua(Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Germplasm Resources Protection and Utilization Platform,Nanjing 210014,China;Jiangsu Key Laboratory of Crop and Livestock Integration,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院畜牧研究所,江苏南京210014 [2]江苏省农业种质资源保护与利用平台,江苏南京210014 [3]农业农村部种养结合重点实验室,江苏南京210014

出　　处：《江苏农业学报》2023年第4期1036-1042,共7页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省种业振兴揭榜挂帅项目[JBGS(2021)099];扬州市重点研发项目(现代农业)(YZ2021037);国家生猪产业技术体系项目(CARS-PIG-35);江苏省农业重大新品种创制项目(PZCZ201733)。

摘　　要：本研究利用RT-PCR检测LncRNA-NEAT1基因表达在细胞中的亚定位,采用5′RACE获得LncRNA-NEAT1基因的转录起始位点;利用CRISPOR软件对-300~0 bp区域的LncRNA-NEAT1启动子序列设计sgRNA并构建到px330载体,通过转染细胞与T7E1酶切验证sgRNA效率;利用酶切和亚克隆方法将dCas9-KRAB-BSD片段替换px330载体的Cas9序列,形成重组px330-dCas9-KRAB载体;将验证有效的sgRNA构建到px330-dCas9-KRAB载体,形成px330-sgRNA-dCas9-KRAB载体。添加不同质量浓度的Blasticidin S处理细胞,以最小致死质量浓度来确定筛选质量浓度。转染px330-sgRNA-dCas9-KRAB载体并用Blasticidin S筛选细胞7 d后进行LncRNA-NEAT1基因的表达检测,同时对sgRNA在LncRNA-NEAT1基因启动子上的结合位点进行Sanger测序,以进一步验证上述结合位点是否发生切割。结果显示LncRNA-NEAT1主要表达于细胞核,而在细胞质中几乎不表达。5′RACE获得了LncRNA-NEAT15′端大约270 bp的序列。qPCR检测结果显示,与对照组相比,sgRNA1和sgRNA2能够显著抑制LncRNA-NEAT1的表达(P<0.05)。Sanger测序结果表明sgRNA1和sgRNA2所在的位点并没有发生碱基缺失和插入。研究结果为后续进一步研究LncRNA-NEAT1在先天性免疫反应中的功能奠定了基础。In this study,the sub-localization of LncRNA-NEAT1 in cells was detected by RT-PCR.And the transcription initiation site of LncRNA-NEAT1 gene was obtained by 5′RACE.The CRISPOR software was used to design the sgRNA in the promoter sequence of LncRNA-NEAT1 within-300~0 bp region.The selected sgRNA was constructed into px330,and the efficiency of sgRNA was verified by transfected cells and T7E1 digestion.Cas9 fragment in the px330 vector was replaced by the dCas9-KRAB-BSD fragment using enzyme digestion and subcloning to form a recombinant px330-dCas9-KRAB vector.It was verified that the effective sgRNA was constructed into the px330-dCas9-KRAB vector to form the px330-sgRNA-dCas9-KRAB vector.Cells were treated by adding different concentrations of Blasticidin S.And the screening concentration was determined by the minimum lethal concentration.The px330-sgRNA-dCas9-KRAB vector was transfected into the cells.After seven days of screening cells with Blasticidin S,the expression of LncRNA-NEAT1 gene was detected.Meanwhile,the binding site of sgRNA on the LncRNA-NEAT1 gene promoter was subjected to Sanger sequencing to further verify whether the above binding site was cleaved.The results showed that LncRNA-NEAT1 was mainly expressed in the nucleus,but hardly in the cytoplasm.The 5′RACE obtained a sequence of approximately 270 bp at the 5′end of LncRNA-NEAT1.The qPCR results showed that compared with the control group,sgRNA1 and sgRNA2 could significantly inhibit the expression of LncRNA-NEAT1(P<0.05).Sanger sequencing results showed that there were no deletions and insertions at the sites where sgRNA1 and sgRNA2 were located.The results laid the foundation for further research on the function of LncRNA-NEAT1 in the innate immune response.

关 键 词：CRISPR dCas9-KRAB LncRNA-NEAT1基因 猪 基因沉默 

分 类 号：S852.28[农业科学—基础兽医学]