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作物学报 ›› 2021, Vol. 47 ›› Issue (3): 427-437.doi: 10.3724/SP.J.1006.2021.04178

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究

周冠彤(), 雷建峰, 代培红, 刘超, 李月, 刘晓东*()   

  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
  • 收稿日期:2020-08-05 接受日期:2020-10-14 出版日期:2021-03-12 网络出版日期:2020-10-28
  • 通讯作者: 刘晓东
  • 作者简介:E-mail: 601930485@qq.com
  • 基金资助:
    国家自然科学基金项目(31660433);本新疆农业大学研究生科研创新项目资助(XJAUGRI2017003)

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

ZHOU Guan-Tong(), LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong*()   

  1. College of Agriculture, Xinjiang Agricultural University, Engineering Research Centre of Cotton of Ministry of Education, Urumqi 830052, Xinjiang, China
  • Received:2020-08-05 Accepted:2020-10-14 Published:2021-03-12 Published online:2020-10-28
  • Contact: LIU Xiao-Dong
  • Supported by:
    National Natural Science Foundation of China(31660433);Xinjiang Agricultural University Postgraduate Research and Innovation Project(XJAUGRI2017003)

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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。

关键词: 棉花, 瞬时转化, CRISPR/Cas9, 基因组编辑

Abstract:

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-sgRNA- CLCrVA, 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-CLCrVA vector was uncovered. The types of mutations included base substitution, base deletion and base insertion. The results indicated that the strategy of using Cas9 transgenic plants as transformation recipients can efficiently and truly verify the effectiveness of sgRNA, which eliminated false negative results due to low transformation efficiency, and the strategy of using virus as vectors to deliver sgRNA was more efficient and accurate. The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.

Key words: cotton, transient transformation, CRISPR/Cas9, genome editing

表1

本研究中使用的引物序列"

引物名称
Primer name		 上游引物序列
Forward sequence (5'-3')		 下游引物序列
Reverse sequence (5'-3')
GhU6-5P-MAPKKK2-sg AAGGGTTCCCAGCTGACATA TATGTCAGCTGGGAACCCTT
GhU6-5P-AE-sg GAGTTTGGAGGGCTTACAAT ATTGTAAGCCCTCCAAACTC
GhU6-5P-2-PDS-sg GAAGCGAGAGATGTTCTAGG CCTAGAACATCTCTCGCTTC
GhU6-5P-2-CLA1-sg TATGCTCGCGGAATGATCAG CTGATCATTCCGCGAGCATA
Test MAPKKK2-sg CCATGTCGTAGCTTATAAAGG TCATTTACCTTCTCTTCCCAG
Test AE-sg AGACTTGTTTCAATGGACTC AATAAGCTGACAGCAGTTGG
Test PDS-sg TGCATGATCCATCACTCAAGTTT GAACGAAAGGCCCTTTCTTTC
Test CLA1-sg GGATCTGAAAGGTGAAAGGAATC TACCGTGATACTTGTCAGCAGCT

图1

基因编辑载体的酶切鉴定 A: 1、2分别为GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::MAPKKK2-sgRNA-Cas9. B: 1、2分别为GhU6-5P::AE-sgRNA-1300和GhU6-5P::AE-sgRNA-Cas9. C: 1~4分别为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. M: 2K plus II DNA marker。"

图2

GhU6-5P::MAPKKK2-sgRNA靶向突变的检测 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-Cas9单株样品基因组PCR扩增产物酶切后结果。B: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-1300单株样品基因组PCR扩增产物酶切后结果。C: GhMAPKKK2-sgRNA靶序列及突变PCR产物的测序。M1发生在D亚组的GhMAPKKK2, M2、M3发生在A亚组GhMAPKKK2。D: 突变PCR产物克隆的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

图3

GhU6-5P::AE-sgRNA靶向突变的检测 A: M: 2K plus II DNA marker; 12、11分别为对照载体基因组PCR扩增产物酶切前后结果; 3~12为转化GhU6-5P::AE-sgRNA-Cas9单株样品基因组PCR扩增产物酶切后结果。B: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~12为转化GhU6-5P::AE-sgRNA-1300单株样品基因组PCR扩增产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图4

GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~6为转化GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: M: 2K plus II DNA marker; 1~10为单克隆菌液PCR产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图5

GhU6-5P-2::AE-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~7为转化GhU6-5P-2::AE-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: M: 2K plus II DNA marker; 1~10为单克隆菌液PCR产物酶切后结果。PCR/RE表示PCR扩增产物酶切分析。"

图6

GhU6-5P-2::PDS-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切后和酶切前结果。B: M: 2K plus II DNA marker; 3~5为转化GhU6-5P-2::PDS-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: GhPDS-sgRNA靶序列及突变PCR产物的测序。M1、M2发生在A亚组的GhPDS, M3发生在D亚组的GhPDS。D: 突变PCR产物的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

图7

GhU6-5P-2::CLA1-sgRNA-CLCrVA部分编辑效应测序检测结果 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果。B: M: 2K plus II DNA marker; 3~5为转化GhU6-5P-2::CLA1-sgRNA-CLCrVA单株样品基因组PCR扩增产物酶切后结果。C: GhCLA1-sgRNA靶序列及突变PCR产物的测序。M1, M3发生在D亚组的GhCLA1, M2发生在A亚组的GhCLA1。D: 突变PCR产物的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。"

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