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Efficient targeted genome modification in Maize Using CRISPR/Cas9 System

Journal of Genetics and Genomics 2016年第1期43卷 37-43页

作者： Chao Feng Jing Yuan Rui Wang Yang Liu James A. Birchler Fangpu Han State Key Laboratory of Plant Cell and Chromosome Engineering Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100101 China University of Chinese Academy of Sciences Beijing 100049 China Division of Biological Sciences University of Missouri Columbia MO 65211-7400 USA

CRISPR （clustered regularly interspaced short palindromic repeats）/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel （insertion and deletion） mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA- Cas9 constructs were transformed into protoplasts followed by sequencmg the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM （protospacer adjacent motif） or having more than two mismatches. Together. our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.

关键词： CRISPR/Cas9 targeted genome modification Heterochromatic region Maize

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High-efficiency targeted modification of plant genomes using TALENs strategy

High-efficiency targeted modification of plant genomes using...

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从植物科学到农业发展——2012全国植物生物学大会

作者： Qiwei Shan~1,Yanpeng Wang~1,Zhen Liang~1,Jun Li,Yi Zhang~1,Kang Zhang~1,Jinxing Liu~1, Kunling Chen~1,Yong Zhang~2,Caixia Gao~(1*) 1 Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101, China. 2 School of Life Science and Technology,University of Electronic Science and Technology of China, Chengdu 610054,China.

Recent advances in high-efficiency methods for targeted genome modification have equipped plant biologists with another powerful genetic tool to facilitate understanding gene functions and expedite translational research to develop crop varieties with higher productivity,enhanced nutrition and lower input *** genome modifications often require targeted cleavage of specific chromosomal sequences,which generates double strand DNA breaks(DSBs) and activates endogenous DNA repair *** have recently developed platforms to design and generate highly specific and active TALE nucleases(TALENs),for recognizing and cleaving desired DNA *** reagents and delivery methods have been optimized to achieve highly efficient targeted mutagenesis in a number of plant species,including rice,Brachypodium,wheat and *** have assembled specific TALENs designed to modify more than 20 genes from rice, Brachypodium and wheat,*** 35.2%of the transgenic rice resistant callus lines, TALENs introduced targeted mutations were *** high efficiencies make it possible to recover genome modifications even without enrichment regimes,As the results, our technology opens the door to make targeted genome modification of plants a routine practice and promises to facilitate creation of new crop varieties to meet burgeoning agricultural ***,the potential impact of targeted genome engineering technology on regulatory decisions will also be presented.

关键词： TALENs targeted genome modification

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