CRISPR-Cas9系统定向编辑TCR基因的sgRNA筛选

Selection of sgRNA for Efficient Editing of TCR Gene by CRISPR-Cas9 System

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.

To establish a TCR-targeted CRISPR-Cas9 system for study of TCR functions, the CRISPR-Cas-sgRNA constructs targeting TRBC were made based on pX458 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 se-quencing. The fragment exhibiting overlapping peaks in target sequence was subject to T-A cloning. The se-quencing results were then analyzed to confirm the occurrence of indel and calculate the editing efficiency. The results showed that three pX458-sgRNA constructs （N1、 N2、 S1） were made. The transfection efficien-cies 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.＆amp;nbsp;Considering the 24. 2% transfection efficiency, the indel efficiency induced by the sgRNA-S1 is approximate 83%. CRISPR-Cas-sgRNA constructs targeting TRBC were successfully made and a type of sgRNA has been identified for high-efficiency genome editing.