CRISPR/Cas9介导的绵羊示踪脐带间充质干细胞系的建立

CRISPR/Cas9 Mediated Exogenous Gene Knock-in at ROSA26 Locus in Sheep Umbilical Cord Mesenchymal Stem Cells

【目的】利用CRISPR/Cas9技术建立绵羊示踪脐带间充质干细胞系,为间充质干细胞的临床治疗与分化机制研究奠定基础。【方法】根据绵羊ROSA26的基因组序列,利用在线工具ZiFiT Targeter Version 4.2设计合成3对引物,利用点突变法,以px330质粒为模板分别进行PCR,DpnⅠ去除质粒DNA后,PCR产物自身环化,酶切测序鉴定,构建以绵羊Rosa26为靶标基因的sgRNA/Cas9载体,构建的质粒含Cas9和向导RNA(single-guide RNA,sgRNA)表达盒,由U6启动子驱动表达。将上述载体分别利用脂质体转染绵羊脐带间充质干细胞(sUMSCs),提取其基因组PCR后进行T7E1酶切,琼脂糖电泳分析条带灰度以检测载体编辑活性。根据sgRNA序列在绵羊ROSA26靶位点的上下游设计并合成左、右同源臂扩增引物,提取绵羊全基因组为模板分别进行PCR扩增得到左右同源臂,回收纯化后分别与pMD19-Simple连接,酶切测序鉴定获得左、右同源臂重组质粒。根据PCR引入的酶切位点,将左同源臂质粒和Donor表达载体DC-DON-SH02ROSA26进行酶切连接,鉴定获得左臂重组打靶载体,使用相同方法将右同源臂质粒连接到左臂打靶载体上,鉴定获得Donor打靶载体,载体携带嘌呤霉素抗性基因和绿色荧光蛋白(GFP)报告基因。在生长良好的sUMSCs中加入不同浓度的嘌呤霉素,观察细胞存活时间,确定最佳抗性筛选浓度和时间。利用脂质体共转sgRNA/Cas9载体和Donor载体到绵羊间充质干细胞,在其ROSA26位点切割DNA双链,在DNA断裂处通过同源重组方式引入报告基因,转染48 h后进行嘌呤霉素抗性筛选,筛选结束后更换正常培养基继续培养,观察绿色荧光的表达并提取阳性细胞基因组,针对ROSA26位点设计上下游两对引物对其进行PCR检测其整合情况。【结果】(1)针对绵羊Rosa26位点设计3对PCR引物,利用点突变法将sgRNA克隆至px330的BbsⅠ酶切位点上,成功构建sgRNA/Cas9载体px330-sgRNA1/2/3,将其分别转染sUMSCs,T7E1酶切结果表明px330-sgRNA2/3出现脱靶现象,未在靶位点发生编辑,px330-sgRNA1的编辑效率最高,约为20%;(2)基于sgRNA1,PCR法获得打靶载体的左、右同源臂,经一系列分子生物学方法先后连接到载体DC-DON-SH02ROSA26上,经酶切和PCR鉴定,成功获得绵羊ROSA26位点的重组载体sROSA26-HA;(3)筛选得到sUMSCs最佳抗性浓度时间,利用Lipofectamine2000共转染sgRNA/Cas9载体和Donor重组载体到sUMSCs,1.5μg·mL^-1嘌呤霉素筛选15d至对照组细胞全部死亡,获得的阳性克隆并扩大培养。显微镜下可观察到明显的绿色荧光,且与对照组相比,阳性细胞克隆的基因组PCR均检测到特异条带,表明sUMSCs的ROSA26位点发生同源重组,GFP基因被成功敲入到基因组中并能正常表达,该细胞可以用于动物疾病模型中追踪sUMSCs的去向和分化方向的研究。【结论】成功利用CRISPR/Cas9系统在sUMSCs内实现外源GFP基因的定点敲入,获得绵羊示踪脐带间充质干细胞系,为间充质干细胞进一步的临床转化奠定了基础。

【Objective】The use of CRISPR/Cas9 system to establish a sheep tracing umbilical cord mesenchymal stem cell line lays the foundation for the clinical therapy and mechanism of MSCs.【Method】Three single guide RNAs(sgRNA)were designed and synthesized for the ROSA26 locus of sheep by using the online tool ZiFiT Targeter Version 4.2,PCR was performed using the point mutation method with the px330 plasmid as a template.The PCR product was circularized after the plasmid DNA was removed by Dpn I,the sg RNA/Cas9 vector targeting s ROSA26 was constructed after enzyme digestion and sequencing identification.The constructed plasmid contained Cas9 and sgRNA expression cassettes,which were driven by the U6 promoter.Because of the off-target effect of CRISPR/Cas9 system,the vector was transfected into sUMSCs,after extraction of its genome for PCR,T7 E1 enzyme digestion.Then analyzing the gray levels of the bands to detect the vector editing activity.The sgRNA/Cas9 vector can cuts the double-stranded DNA at the target site.In order to knock in the reporter gene,the Donor vector needs to repair the sequence on the donor DNA by homologous recombination at the DNA break to insert target sequence.Based on the sgRNA sequence,the left and right homology arm amplification primers were designed and synthesized at the upstream and downstream of the sROSA26 locus.Using sheep’s whole genome as a template for PCR amplification to obtain left and right homology arms.After recovery and purification,recombinant with pMD19-Simple to obtain left and right homologous recombinant plasmids.The left and right homology arm plasmids were ligated with the Donor expression vector DC-DON-SH02 ROSA26 to obtain the left arm recombinant targeting vector.The homologous recombination vector carrying green fluorescent protein(GFP)and puromycin resistance gene was constructed.The survival time of cells with different concentrations of puromycin was observed in well-growing sUMSCs to determine the optimal concentration and time of resistance screening.In sUMSCs,sgRNA/Cas9 vector and Donor vector were co-transfected by liposome method.Puromycin resistance screening was performed 48 hours after transfection.After the screening,cells need to be replaced with normal media and continue to expand.Observing the expression of GFP and extracting the positive cell genome,designing two pairs of upstream and downstream primers for ROSA26 site for PCR to detect its editing status.【Result】(1)Three sgRNA primers were designed for the sheep ROSA26 locus.The expression vector px330-sgRNA1/2/3 were successfully constructed by point mutation method and the vectors were transfected into sheep umbilical cord mesenchymal stem cells.T7 E1 enzyme assay results indicated that the editing efficiency of px330-sgRNA1 was the highest,about 20%.(2)Based on sgRNA1,the left and right homology arms of the targeting vector were obtained by PCR,and the recombinant vector(sROSA26-HA)of sheep ROSA26 locus was successfully obtained by a series of molecular biological methods;(3)px330-sgRNA1 and sROSA26-HA were co-transfected into sheep umbilical cord mesenchymal stem cells using Lipofectamine2000,and positive colonies could express green fluorescent protein were obtained after 1.5μg·mL^-1 puromycin selection for 15 days.PCR detection of one clone indicated that the targeting vector had been integrated into the genome.【Conclusion】The exogenous GFP gene could successfully integrated in specific locus of sheep umbilical cord mesenchymal stem cells using the CRISPR/Cas9 system,and these cells can be used to trace the position and differentiated cell type of MSCs.This research laid a foundation for further clinical transformation of mesenchymal stem cells.