集美大学学报（自然科学版）Issue(4)：265-270,6.
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
摘要
Abstract
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. 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.关键词
T细胞抗原受体/靶向/CRISPR/基因组/编辑Key words
TCR/targeting/clustered regularly interspaced short palindromic repeats(CRISPR)/genome/editing分类
医药卫生引用本文复制引用
邵红伟,陈辉,彭鑫,徐畅,张广献,黄树林..CRISPR-Cas9系统定向编辑TCR基因的sgRNA筛选[J].集美大学学报（自然科学版）,2015,(4):265-270,6.基金项目
国家自然科学基金资助项目(31100664,31300737,81303292) （31100664,31300737,81303292）
