CRISPR-Cas9系统定向编辑TCR基因的sgRNA筛选-《集美大学学报（自然科学版）》

文章信息/Info

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

作者:: 邵红伟1; 2; 陈辉1; 2; 彭鑫1; 2; 徐畅1; 2; 张广献3; 黄树林1; 2; （1.广东药学院生命科学与生物制药学院，广东广州510006；2.广东省生物技术候选药物研究重点实验室，广东广州510006；3.广州中医药大学基础医学院，广东广州 510006）

Author(s):: SHAO Hong-wei1; 2; CHEN Hui1; 2; PENG Xin1; 2; XU Chang1; 2; ZHANG Guang-xian3; HUANG Shu-lin1; 2; (1.School of Biosciences and Biopharmaceutics，Guangdong Pharmaceutical University，Guangzhou 510006，China；2.Guangdong Province Key Laboratory for Biotechnology Drug Candidate，Guangzhou 510006，China;3.School of Basic Medical Sciences，Guangzhou University of Chinese Medicine，Guangzhou 510006，China)

Keywords:: TCR; targeting; clustered regularly interspaced short palindromic repeats(CRISPR); genome; editing

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pX458质粒构建靶向TCR基因β链C区的CRISPR-Cas-sgRNA质粒，将其转染HepG2细胞系，用流式细胞术检测转染效率；转染48 h后提取HepG2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pX458-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-Cas-sgRNA质粒，并鉴定出基因编辑效率较高的一种sgRNA序列.

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 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 pX458-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 approximate 83%.CRISPR-Cas-sgRNA constructs targeting TRBC were successfully made and a type of sgRNA has been identified for high-efficiency genome editing.