基于CRISPR/Cas9技术的同源修复载体构建  

Construction of Homologous Repair Vector Based on CRISPR/Cas9 Technology

作　　者：马铭赛 徐锡荣 布梁灏 黄嘉玲 欧阳乐军 李莉梅 刘智超 孙同川 梁楚炎 Ma Mingsai;Xu Xirong;Bu Lianghao;Huang Jialing;Ouyang Lejun;Li Limei;Liu Zhichao;Sun Tongchuan;Liang Chuyan(College of Biological and Food Engineering,Guangdong University of Petrochemical Technology,Maoming,525000;The Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges&Universities under the Department of Education of Xinjiang Uygur Autonomous Region,College of Life and Geographic Sciences,Kashi University,Kashi,844000)

机构地区：[1]广东石油化工学院生物与食品工程学院,茂名525000 [2]喀什大学生命与地理科学学院,叶尔羌绿洲生态与生物资源研究高校重点实验室,喀什844000

出　　处：《基因组学与应用生物学》2020年第10期4680-4685,共6页Genomics and Applied Biology

基　　金：广东省自然科学基金(2017A030307017);广东省大学生攀登计划项目(pdjh2019b0323);广东科技计划项目(2017A030303087)共同资助。

摘　　要：为探索利用CRISPR/Cas9系统在拟南芥中进行大片段基因敲除并产生回复突变的可行性及其工作效率进一步精准验证靶基因在植物的生长发育中以及响应逆境胁迫中所起的作用。本研究利用同源重组法将sgRNA和编码Cas9的序列快速连接构建表达载体,同时在载体两端装载缺失片段的同源臂以提供同源重组修复时所需的模板。结果显示,敲除ASⅠ基因的表达载体pHDE-ASⅠ重组子阳性率为100%,同源修复载体pHDE-ASⅠ-EcoRⅠ-mCherry为90.9%,且经测序验证,利用同源重组法所构建的表达载体序列无突变,与原序列高度一致从而实现了同源修复载体的构建。本研究为精准验证靶基因的功能提供了技术指导。To explore the feasibility and efficiency of using CRISPR/Cas9 system to carry out large fragment gene knockout and generate revertive mutation in A rabidopsis thaliana,and to accurate verification of the role of target genes in plant growth and development and in response to stress furtherly,Homologous recombination method was used to rapidly connect sgRNA and Cas9 coding sequences to construct expression vectors in this study,and homologous arms of missing fragments were loaded at both ends of the vectors to provide templates for homologous recombination repair.The results showed that the recombinant positivity rates of pHDE-ASⅠ-mCherry vector was 100%and the recombinant positivity rates of pHDE-ASⅠ-EcoRⅠ-mCherry vector was90.9%,respectively.After sequencing verification,the expression vector sequences constructed by homologous recombination method had no mutation,and highly consistent with the original sequence,which realized the construction of homologous chromosome repair vector.It would provide a technical indication for the accurate verification of the function of the target gene.

关 键 词：CRISPR/Cas9 同源重组 载体构建 基因编辑 

分 类 号：Q78[生物学—分子生物学]