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

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。

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

Objective:To clone the scaffold guide RNA expression vectors targeting Wnt pathway and to verify the efficacy ofthese plasmids in fine-tuning Wnt pathway using an HEK293 cell model.Methods:The negative regulators of Wnt pathway were selectedin this study. Guide RNAs targeting the promoter region of these genes were designed and corresponding DNA oligos were synthesizedfor cloning into lenti-sgRNA-Ms2-zeo. The plasmid construction were done by annealing the two complementary DNA sequences beforeinsertion into lenti-sgRNA-Ms2-zeo vectors with the enzyme BsmBI. 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 thetarget genes were analyzed by qPCR assay.Results:Four genes were selected in this study, all of which were well-established negativeregulators of Wnt pathway. Two guide RNAs recognizing the promoter of each gene were designed and corresponding plasmids expressingthe gRNA fused with a MS2 scaffold were constructed. Sequencing results indicated that all the clones were correctly done. 4 kindsof plasmids were randomly selected for further gene activation efficiency analysis. As expected, the gRNA expression vectors could significantlyincrease the target genes when co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine.Conclusion:The plasmids that expressed gRNAs targeting the known Wnt negative regulators were successfully constructed, which could fine-tunethe Wnt pathway by increase the target gene expression.