利用CRISPR/Cas9技术缺失MDV分离株meq基因的研究

Study on Deletion of meq Gene of MDV Isolate by CRISPR/Cas9 System

鸡马立克氏病病毒(MDV)是一种能够引起鸡肿瘤的疱疹病毒,具有严格的细胞结合特性,对其基因组编辑较为困难。研究利用CRISPR/Cas9技术对中国MDV分离株基因组中的meq基因进行缺失。依据中国MDV分离株BS/15株的meq基因序列,设计了8条靶向meq基因的小向导RNA(sgRNA),分别克隆到pX330质粒中,构建表达靶向meq基因sgRNA的质粒pMeqsgRNA。通过pMeq-sgRNA转染、测序分析方法初步评价sgRNA切割效果,选择了两条适合的sgRNA。在鸡胚成纤维细胞(CEF)上同时转染上述筛选的2个pMeq-sgRNA质粒,接种MDV分离株BS/15株,通过PCR检测筛选meq基因缺失克隆株,成功克隆到一株meq基因缺失株。序列分析证明获得的MDV重组毒株确定meq基因序列已被敲除。体外试验证明该MDV重组毒株与亲本毒株在CEF上具有相似的复制能力。研究提供了一种简单、快捷的编辑MDV基因的方法,为MDV功能基因、分子进化和新型基因工程疫苗的研究提供了技术支持。

Marek′s disease virus(MDV)is a herpesvirus that causes tumors in chickens.It has strict cell-associated properties and is difficult to edit its genome.This study utilised CRISPR/Cas9 system to delete the meq gene of Chinese MDV isolates.According to the meq gene sequence of Chinese MDV isolate BS/15 strain,eight small guide RNAs(sgRNAs)targeting meq gene were designed and cloned into pX330 plasmid,and the high efficiency of sgRNA was initially screened by sgRNA transfection and sequencing analysis.Two sgRNAs with the highest efficiency were selected and transfected into chicken embryo fibroblast(CEF).The meq gene-deleted strain was screened by PCR detection.Finally,a meq gene-deleted strain was successfully cloned.Sequence analysis revealed that the obtained MDV recombinant strain had been knocked out the meq gene at the expected cleavage site.It had been proved that the meq-deleted strain and the parent strain had similar replication ability on CEF.This study provided a simple and rapid method for editing MDV genes,providing technical support for the study of MDV functional genes,molecular evolution and novel genetic engineering vaccines.