【摘要】 为构建靶向T细胞抗原受体(T Cell Receptor,TCR)基因的CRISPR-Cas9基因组编辑系统,基于p X458质粒构建靶向TCR基因β链C区的CRISPR-Cas-sgRNA质粒,将其转染Hep G2细胞系,用流式细胞术检测转染效率;转染48 h后提取Hep G2细胞基因组DNA,扩增含有编辑位点的片段,测序分析该片段的峰图改变;对出现双峰的扩增片段做T-A克隆后测序分析,确定基因编辑发生的位置,并结合转染效率计算基因编辑效率.结果表明,成功构建含有3种sgRNA序列(N1、N2、S1)的p X458-sgRNA质粒,其转染效率分别为38.5%(N1)、39.7%(N2)和24.2%(S1);基因组PCR产物测序分析发现,S1组扩增片段在打靶位置出现杂峰;T-A克隆测序发现,20克隆有4个发生了基因编辑(20%),结合转染效率(24.2%)可知,编辑效率约为83%.可见,本文成功构建靶向TCR基因的CRISPR-CassgRNA质粒,并鉴定出基因编辑效率较高的一种sgRNA序列.更多还原

【Abstract】 To establish a TCR-targeted CRISPR-Cas9 system for study of TCR functions,the CRISPRCas-sgRNA constructs targeting TRBC were made based on p X458 vector and transferred into HepG2. The transfection efficiencies were detected by flow cytometry( FCM) after 48 h. Subsequently,the genomic DNA of HepG2 was extracted and a fragment covering target sequence was amplified by PCR and analyzed by sequencing. The fragment exhibiting overlapping peaks in target sequence was subject to T-A cloning. The sequencing results were then analyzed to confirm the occurrence of indel and calculate the editing efficiency.The results showed that three p X458-sgRNA constructs( N1、N2、S1) were made. The transfection efficiencies were 38. 5%( N1),39. 7%( N2) and 24. 2%( S1),respectively. Sequencing results of S1 fragment exhibited overlapping peaks. After cloning and sequencing,4 of 20 S1 clones showed sequence alteration.Considering the 24. 2% transfection efficiency,the indel efficiency induced by the sgRNA-S1 is approximate83%. CRISPR-Cas-sgRNA constructs targeting TRBC were successfully made and a type of sgRNA has been identified for high-efficiency genome editing.更多还原