基于CRISPR-Cas9系统的HSV-1基因治疗载体的快速构建

Rapid Construction of HSV-1 Vectors for Gene Therapy Based on CRISPR/Cas9-mediated Viral Genome Editing

基因改造的1型单纯疱疹病毒(HSV-1)载体在肿瘤溶瘤病毒治疗及基因转导中具有广泛的应用前景。本研究报道一种基于CRISPR-Cas9系统的高效快速的重组HSV-1载体构建方法。首先,双顺反表达靶点g DNA和Cas9核酸酶的基因编辑质粒与同源重组模板质粒共转染Vero细胞后,用亲本株感染细胞;然后,Cas9对胞内病毒基因组定点切割,诱导外源基因同源重组,修复至病毒基因组指定位点。通过PCR、Western印迹、免疫荧光等方法证明,相比于传统自发同源重组的构建方法,该方法能显著提升病毒重组率(4.1%vs 1.1%)。同时,本研究建立了一种新的单克隆病毒纯化方案,简化了阳性病毒筛选步骤。本研究结果提供了一种高效快速的重组HSV-1构建方法,这对于HSV-1相关基因治疗及其病理机制研究将具有重要意义。

The genetically modified HSV-1 virus serves as promising vectors for oncolytic tumor therapy and in vivo gene transduction. Herein we report an efficient and rapid HSV-1 gene editing method based on the CRISPR-Cas9 system. During replication of the HSV-1 1716 strain in Vero ceils, pre-transfected CRISPR-Cas9 systems could direct a precise cleavage in the target region and induce gene replacement between the target region and the import sequence mediated by homology-directed repair. PCR, Western blotting and immunofluorescence analyses showed that compared with the classical spontaneous homologous recombination method, this method significantly increased the recombination rate （4. 1% vs 1.1% ）. Meanwhile, we developed a novel isolation platform for desired recombinant viruses using the single virus plaque formation method. Together, we provide an improved method for construction HSV-1 vectors, which will facilitate the development of related researches on the mechanisms of pathogenesis and HSV-l-mediated gene therapy.