CRISPR/Cas9技术联合AAV对Rosa-mTmG小鼠的MEF细胞进行编辑

Editing of MEF Cells From Rosa-mTmG Mice by Using CRISPR/Cas9 in Combination with AAV

目前,基因编辑技术已被广泛用于生物医学研究,本研究利用AAV(Adeno-Associated Virus)递送CRISPR/Cas9系统,实现高效率的基因编辑,并对特定组织细胞中的基因编辑进行了初步探索.我们首先采用Rosa-mTmG转基因小鼠来研究CRISPR/Cas9在小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblast,MEF)中的编辑效率;接着,我们构建了能够被AAV包装的SaCas9系统,并通过瞬转以及AAV介导递送的方式检测其基因编辑效率;最后,我们还构建了含有心肌特异启动子的cTNT-PX601-sgRNA以备后续研究.结果发现,与瞬转相比,AAV介导CMV-PX601-sgRNA在体外能够极大地提高细胞的基因编辑效率.另外,cTNT-PX601-sgRNA能够在心肌细胞中特异表达.我们的结果表明,利用AAV介导的CRISPR/Cas9系统在体外可以实现高效地基因编辑,为靶向修复基因缺陷疾病,特别是特定组织器官中的基因缺陷,提供了研究工具和实验依据.

At present,gene editing technology has been widely used in biomedical research.This study uses AAV to deliver the CRISPR/Cas9 system for efficient gene editing,and the gene editing in specific tissues and cells was preliminarily explored.First,the Rosa-mTmG transgenic mouse was used to study the editing efficiency of CRISPR/Cas9 in mouse embryonic fibroblasts.Then,we constructed AAV system containing the SaCas9 gene,and detected its efficiency of gene editing by transient transfection and AAV-mediated manners.Finally,we constructed a plasmid named cTNT-PX601-sgRNA containing a cardiac specific promoter for subsequent studies.It was found that AAV-mediated CMV-PX601-sgRNA can greatly improve the efficiency of gene editing compared with transient transfection.In addition,cTNT-PX601-sgRNA can be specifically expressed in cardiomyocytes.Our results indicate that the AAV-mediated CRISPR/Cas9 system can achieve efficient gene editing in vitro,and it provides research tools and experimental evidences for targeted repair of gene-deficient diseases,especially gene defects in specific tissues and organs.