棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究  

Efficient screening system of effective sgRNA for cotton CRISPR/Cas9 gene editing

作　　者：周冠彤 雷建峰[1] 代培红[1] 刘超[1] 李月[1] 刘晓东[1] ZHOU Guan-Tong;LEI Jian-Feng;DAI Pei-Hong;LIU Chao;LI Yue;LIU Xiao-Dong(College of Agriculture,Xinjiang Agricultural University,Engineering Research Centre of Cotton of Ministry of Education,Urumqi 830052,Xinjiang,China)

机构地区：[1]新疆农业大学农学院/棉花教育部工程研究中心,新疆乌鲁木齐830052

出　　处：《作物学报》2021年第3期427-437,共11页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31660433);新疆农业大学研究生科研创新项目(XJAUGRI2017003)资助。

摘　　要：单向导RNA(sgRNA)是CRISPR/Cas9基因组编辑技术体系的重要元件之一。然而研究显示,很多sgRNA不能有效工作,因此需要对多个设计的候选sgRNA进行筛选,以验证它们的有效性。早期对sgRNA有效性的验证采用的是完整编辑载体瞬时转化原生质体或者叶片的方法。这些方法费时费力,成功率不高,尤其是对于原生质体制备效率比较低的棉花。本研究针对GhMAPKKK2和GhAE基因分别设计靶序列,构建了只转录sgRNA的载体:GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::AE-sgRNA-1300,并通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片;与此同时,构建了对应完整的CRISPR/Cas9基因组编辑载体:GhU6-5P::MAPKKK2-sgRNA-Cas9和GhU6-5P::AE-sgRNA-Cas9,并通过农杆菌注射YZ-1野生型棉花植株的叶片。另外,针对GhPDS、GhCLA1、GhMAPKKK2和GhAE基因分别设计靶序列并构建了GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1-sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA病毒投送载体,通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片。以上试验均以转化对应空载体的植株为对照。对转化后的棉花叶片基因组DNA进行PCR扩增后酶切,并对未完全消化的PCR产物进行克隆测序,结果显示,转化GhU6-5P::AE-sgRNA-1300、GhU6-5P::MAPKKK2-sgRNA-Cas9、GhU6-5P::AE-sgRNA-Cas9载体的棉花植株均未检测到靶基因突变,而转化GhU6-5P::MAPKKK2-sgRNA-1300、GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1-sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA载体的Cas9转基因阳性植株基因序列发生了改变,突变类型包括碱基替换、碱基缺失和碱基插入。表明以Cas9转基因阳性植株为转化受体的策略可以高效真实地验证sgRNA的有效性,排除了因转化效率低而带来的假阴性的结果,且病毒载体投送sgRNA的策略更高效、更准确。该sgRNA高效验证体系的建立,为棉花功能基因组学研究提供了重要的技术基础。Single guide RNA(sgRNA)is one of the important elements of the CRISPR/Cas9 genome editing technology system.However,studies have shown that many sgRNAs cannot work effectively.It is worth screening to verify the effectiveness of multiple design candidate sgRNAs.Instantaneous transformation of protoplasts or leaves with complete editing vectors were used to verification of the effectiveness of sgRNA in the early stage.These methods are time-consuming and laborious,and the success rate is not high,especially for cotton with low efficiency of the protoplasmic system.In this study,target sequences were designed for GhMAPKKK2 and GhAE genes,and two vectors of GhU6-5P::MAPKKK2-sgRNA-1300,GhU6-5P::AE-sgRNA-1300 which transcibed only sgRNA were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium;meanwhile,two corresponding complete CRISPR/Cas9 genome editing vectors of GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9 were constructed and injected YZ-1 wild-type cotton leaves with Agrobacterium.In addition,target sequences were designed for GhPDS,GhCLA1,GhMAPKKK2,and GhAE genes,respectively,and GhU6-5P-2::PDS-sgRNACLCrVA,GhU6-5P-2::CLA1-sgRNA-CLCrVA,GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA virus delivery vectors were constructed and injected YZ-1 Cas9 transgenic cotton plant leaves through Agrobacterium.In the above experiments,the plants transformed with the empty vector were used as controls.The genomic DNA of the transformed cotton leaves was subjected to PCR and enzyme digestion,and the PCR products which were not completely digested were cloned and sequenced.The results showed that no mutation in target gene was detected in the cotton plants transformed with the GhU6-5P::AE-sgRNA-1300,GhU6-5P::MAPKKK2-sgRNA-Cas9 and GhU6-5P::AE-sgRNA-Cas9,and the target genes mutation in the Cas9 transgenic plants transformed with GhU6-5P::MAPKKK2-sgRNA-1300,GhU6-5P-2::PDS-sgRNA-CLCrVA,GhU6-5P-2::CLA1-sgRNA-CLCrVA,GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-C

关 键 词：棉花 瞬时转化 CRISPR/Cas9 基因组编辑 

分 类 号：S562[农业科学—作物学] Q943.2[生物学—植物学]