利用改良的CRISPR/Cas9系统构建猪PPARD载体及效率检测  

Construction and Efficiency Detection of a Porcine PPARD Vector Using Improved CRISPR/Cas9 System

作　　者：吴雅婕 杨跃飞[1] 范博钧 徐磊[1] 鞠辉明[1,2] WU Yajie;YANG Yuefei;FAN Bojun;XU Lei;JU Huiming(College of Veterinary Medicine,Yangzhou University,Yangzhou Jiangsu 225009,China;Jiangsu Collaborative Innovation Center for Animal Epidemics and Zoonoses,Yangzhou Jiangsu 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《广西师范大学学报（自然科学版）》2023年第6期132-138,共7页Journal of Guangxi Normal University:Natural Science Edition

基　　金：国家自然科学基金(31872323);江苏省自然科学基金(BK20201224);江苏高校优势学科建设工程资助(2018);江苏高校品牌专业建设工程资助(TAAP2018-2022)。

摘　　要：为了构建猪PPARD基因编辑载体并进行基因编辑效率检测,本研究利用改良的双位点编辑CRISPR/Cas9载体系统,根据PPARD基因结构和序列特点,设计2个能靶向切割PPARD基因的sgRNA序列,将2个PPARD sgRNA表达盒以串联的形式连接到一个CRISPR/Cas9载体中。将构建的质粒转染PK15细胞,提取各组细胞DNA,PCR扩增突变区域后,通过序列测定在DNA水平检测载体编辑效率。分别提取各组细胞总RNA及细胞总蛋白,利用qRT-PCR及Western blot在基因表达及蛋白表达水平上检测重组载体的基因编辑效率。结果发现,PPARD-2KO组DNA总突变率为45.83%(22/48);与正常对照组相比,PPARD基因编辑组中PPARD mRNA显著下降84%(P<0.01),PPARD蛋白表达量显著下降61%(P<0.01)。本研究利用改良的CRISPR/Cas9载体系统成功构建了高效PPARD基因编辑载体,并且未经药物筛选即可在细胞上实现高效率基因编辑,将大幅提高后续筛选单细胞克隆效率,推进对PPARD基因的功能研究。To construct the porcine PPARD gene editing vector and detect the gene editing efficiency.In this study,two sgRNA sequences that can target the cutting of PPARD gene were designed according to the structure and sequence characteristics of PPARD gene,and the two PPARD sgRNA expression boxes were connected to a CRISPR/Cas9 vector in tandem.After the constructed plasmids were transfected into PK15 cells,the DNA of each group of cells was extracted.The mutant region was amplified by PCR,and the vector editing efficiency was detected at the DNA level by sequence determination.The total RNA and total protein of each group were extracted,and the gene editing efficiency of recombinant vectors was detected by qRT-PCR and Western blot at the level of gene expression and protein expression.The total mutation rate of the PPARD-2KO group was 45.83%(22/48).Compared with the normal control group,the PPARD mRNA in the PPARD gene editing group was significantly decreased by 84%(P<0.01),and the PPARD protein expression was significantly decreased by 61%(P<0.01).The results show that this study successfully constructed a high-efficiency PPARD gene editing vector by using the improved CRISPR/Cas9 vector system,and can achieve high-efficiency gene editing on cells without drug screening,which will greatly improve the efficiency of subsequent screening of single-cell cloning and promote the functional study of PPARD genes.

关 键 词：猪 PPARD CRISPR/Cas9 基因编辑 

分 类 号：Q78[生物学—分子生物学] S828.2[农业科学—畜牧学]