中国农业科学2017，Vol.50Issue(7)：1294-1301,8.DOI:10.3864/j.issn.0578-1752.2017.07.012
香蕉CRISPR/Cas9基因编辑技术体系的建立
Establishment of an Efficient CRISPR/Cas9-Mediated Gene Editing System in Banana
摘要
Abstract
[Objective] The objective of this study is to establish a CRISPR/Cas9-mediated gene editing system in order to find a new way for revealing gene function and breeding of banana.[Method] According to the Musa genomic (A genome) sequence of phytoene desaturase gene 8 (MaPDS),online tools ZiFiT Targeter Version 4.2 was used to determine the appropriate designing of CRISPR/Cas9 target sites,one of the output target sites was selected for designing the sgRNA.The sgRNA and U6a promoter sequences were linked by overlapping PCR to produce U6a-sgRNA expression cassette,then the fragment was cloned into the binary pYLCRISPR/Cas9 multiplex genome targeting vector system based on Golden Gate Cloning,a pYLCRISPR/Cas9 vector carrying sgRNA cassette named pYLCRISPR/Cas9-sgRNA was generated.The plant expression binary vector,containing a Cas9 (Cas9p) expression cassette,driven by the maize ubiquitin promoter (Pubi),and the sgRNA with target sequence of MaPDS was transcribed under the control of the U6a promoter from rice.The constructed plasmid was then transformed into EHA 105 strain of Agrobacterium tumefaciens,which was subsequently used to infect embryogenic cell suspensions (ECS) of banana to obtain resistant plantlets.PCR amplifications were carried out using primer pairs flanking the designed target site.The PCR products were sequenced for detection of mutation.[Result] MaPDS was selected as the target ofCas9 endonuclease,and a sgRNA was designed as the target site for CRIPSR/Cas9.The sgRNA with target sequence was firstly inserted into the sgRNA expression cassette by using overlapping PCR,then the cassette was combined with the pYLCRISPR/Cas9 in a single plant binary vector.The results of identification by enzyme digestion and sequencing showed that the pYLCRISPR/Cas9-sgRNA vector aiming at MaPDS was successfully constructed,and the vector harbors a Cas9 expression cassette driven by Pubi,and a sgRNA expression cassette under the U6a promoter.Using this system and Agrobacterium-mediated transformation,129 independently transformed lines with visible albino,chimerism mutant phenotypes and normal phenotypes were regenerated from ECS after transformation,resistance screening,resistant embryo induction,germination and rooting process.The ratio of albino and chimerism phenotype occurred in transgenic banana was more than 55％ (71 out of 129).Albino phenotype in these transgenic plants indicates the loss of PtoPDS function.To further verify whether the loss of green in the transgenic banana was caused by generation of mutations in the MaPDS by the CRISPR/Cas9 system,33 mutant phenotypes and 14 normal phenotypes from 129 independent transgenic plants were randomly selected for sequencing.The results confirmed that all of these transgenic plants with albino phenotype contained mutants in the MaPDS,insertion (a "A" or "T") and displacement were found in the target sites of MaPDS in the mutants.And it was also found the displacement in downstream of the target sites of MaPDS in the experiments,no mutation was detected in the normal phenotypes of transgenic and control plants.[Conclusion] The CRISPR/Cas9 system was successfully applied to knock out the endogenous phytoene dehydrogenase (PDS) gene in banana.The results demonstrate that the Cas9/sgRNA system can be exploited to targeted mutagenesis and effectively create knockout mutations in banana.关键词
香蕉/CRISPR/Cas9/MaPDS/基因编辑Key words
banana/CRISPR/Cas9/MaPDS/gene editing引用本文复制引用
胡春华,邓贵明,孙晓玄,左存武,李春雨,邝瑞彬,杨乔松,易干军..香蕉CRISPR/Cas9基因编辑技术体系的建立[J].中国农业科学,2017,50(7):1294-1301,8.基金项目
农业部“948”项目(2016-X22)、广州市科技计划项目(201707010154)、国家香蕉产业体系(CARS-32-01)、广东省科技计划项目(2014B070706018,2015A030302046)、农业部物质资源保护项目(16RZZY-13) （2016-X22）
