基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证

Construction and Verification of the Plasmids Fine-tuning Wnt Pathway Based on CRISPR/Cas9 Gene Editing System

目的:构建基于CRISPR/cas9系统调控Wnt信号通路的载体,并在细胞水平验证其调控基因表达的效率。方法:选取Wnt信号中负性调控分子,设计并合成能够表达靶向上述分子gRNA的互补DNA克隆序列,BsmBI限制性内切酶酶切载体后,采用分子克隆的方法将上述序列克隆至目的载体lenti-sgRNA-Ms2-zeo,测序正确的克隆通过Lipofectamine2000与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共同转染入293细胞;转染24h后收集细胞,qRt-PCR检测目的基因的表达。结果:筛选了Wnt信号通路中已知的19个负性调控基因;针对每个基因设计了两对gRNA序列,并构建了能够表达gRNA和MS2融合序列的载体,测序结果显示重组质粒的DNA序列与预期完全相符。随机挑选了4个表达载体与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共转进入细胞,qPCR结果显示构建的目的载体联合lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine载体可以协同促进靶分子表达。结论:本研究成功构建了基于CRISPR/cas9基因编辑系统调控Wnt信号的载体。

Objective： To clone the scaffold guide RNA expression vectors targeting Wnt pathway and to verify the efficacy of these plasmids in fine-tuning Wnt pathway using an HEK293 cell model. Methods： The negative regulators of Wnt pathway were selected in this study. Guide RNAs targeting the promoter region of these genes were designed and corresponding DNA oligos were synthesized for cloning into lenti-sgR NA-Ms2-zeo. The plasmid construction were done by annealing the two complementary DNA sequences before insertion into lenti-sgR NA-Ms2-zeo vectors with the enzyme BsmB I. The acquired clones are confirmed by sequencing. After confirmation, the plasmids were co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine into 293 cells. Expression of the target genes were analyzed by qPCR assay. Results： Four genes were selected in this study, all of which were well-established negative regulators of Wnt pathway. Two guide RNAs recognizing the promoter of each gene were designed and corresponding plasmids expressing the gR NA fused with a MS2 scaffold were constructed. Sequencing results indicated that all the clones were correctly done. 4 kinds of plasmids were randomly selected for further gene activation efficiency analysis. As expected, the gR NA expression vectors could significantly increase the target genes when co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine. Conclusions：The plasmids that expressed gR NAs targeting the known Wnt negative regulators were successfully constructed, which could fine-tune the Wnt pathway by increase the target gene expression.