CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum

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• 作者：Junping Gao (1)
  Genhong Wang (1)
  Sanyuan Ma (1)
  Xiaodong Xie (2)
  Xiangwei Wu (1)
  Xingtan Zhang (2)
  Yuqian Wu (2)
  Ping Zhao (1)
  Qingyou Xia (1)
  
• 关键词：CRISPR/Cas9 system ; Nicotiana tabacum ; Targeted mutagenesis ; Genome editing
• 刊名：Plant Molecular Biology
• 出版年：2015
• 出版时间：January 2015
• 年：2015
• 卷：87
• 期：1-2
• 页码：99-110
• 全文大小：921 KB
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  19. Feng Z
• 作者单位：Junping Gao (1)
  Genhong Wang (1)
  Sanyuan Ma (1)
  Xiaodong Xie (2)
  Xiangwei Wu (1)
  Xingtan Zhang (2)
  Yuqian Wu (2)
  Ping Zhao (1)
  Qingyou Xia (1)
  
  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China
  2. School of Life Science, Chongqing University, Chongqing, 400030, China
  
• ISSN：1573-5028

文摘

Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-0.3?% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8?% for NtPDS gRNA4 and 87.5?% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.