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草业学报 ›› 2023, Vol. 32 ›› Issue (4): 129-141.DOI: 10.11686/cyxb2022180

• 研究论文 • 上一篇    

tRNA-sgRNA/Cas9系统介导多年生黑麦草原生质体的基因编辑

姚佳明(), 郝欢欢, 张敬, 徐彬()   

  1. 南京农业大学草业学院,江苏 南京 210095
  • 收稿日期:2022-04-20 修回日期:2022-06-27 出版日期:2023-04-20 发布日期:2023-01-29
  • 通讯作者: 徐彬
  • 作者简介:E-mail: binxu@njau.edu.cn
    姚佳明(1996-),男,河北保定人,在读硕士。E-mail: 873606461@qq.com
  • 基金资助:
    国家自然科学基金(31971757)

The use of the tRNA-sgRNA/Cas9 system for gene editing in perennial ryegrass protoplasts

Jia-ming YAO(), Huan-huan HAO, Jing ZHANG, Bin XU()   

  1. College of Agro-grassland Science,Nanjing Agricultural University,Nanjing 210095,China
  • Received:2022-04-20 Revised:2022-06-27 Online:2023-04-20 Published:2023-01-29
  • Contact: Bin XU

摘要:

tRNA可以将多个sgRNAs连接起来合并成一个多顺反子基因,再与CRISPR/Cas9表达载体结合形成多顺反子tRNA-sgRNA/Cas9(PTG/Cas9)系统来对多靶点进行基因编辑。该系统已经在水稻中验证其可促进sgRNAs的转录和提高多靶点的编辑效率。因此,为了在多年生黑麦草中实现高效的基因编辑,本研究中,构建了2个带有tRNA的CRISPR中间载体,并提供了一种快速、灵活的PTG/Cas9载体构建方法。为了快速验证PTG/Cas9系统是否可以在多年生黑麦草基因组中发挥功能,用聚乙二醇4000(PEG 4000)介导PTG/Cas9质粒转化多年生黑麦草原生质体,后提取原生质体DNA扩增目的序列检测是否有目标基因被编辑的细胞。试验结果显示,PTG/Cas9系统可用于多年生黑麦草基因编辑,且基因编辑效率约为6.7%。试验结果为多年生黑麦草遗传研究和育种提供了重要的基础。

关键词: CRISPR/Cas9, tRNA, 多基因编辑, 载体, 质粒, 原生质体, 多年生黑麦草

Abstract:

Transfer RNA (tRNA) can link multiple sgRNAs (single-guide RNAs) to form a polycistronic gene, which then combines with a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9) expression vector to form a polycistronic tRNA-sgRNA/Cas9 (PTG/Cas9) system for multiple gene editing. The PTG/Cas9 system has been used to alter sgRNAs transcript levels and improve multi-target editing efficiency in rice (Oryza sativa). To efficiently edit target genes in perennial ryegrass (Lolium perenne), we generated two CRISPR intermediate vectors with tRNAs to provide a fast and flexible PTG/Cas9 vector construction method. To verify whether the PTG/Cas9 system effectively edits genes in the perennial ryegrass genome, we introduced the PTG/Cas9 plasmid into perennial ryegrass protoplasts by PEG 4000-mediated transformation. Then, we extracted DNA from protoplasts and amplified the target sequences to determine whether they had been edited successfully. The gene editing efficiency was about 6.7%. These results show that the PTG/Cas9 system can be used for gene editing in the ryegrass genome, and provide the basis for further genetic research on, and breeding of perennial ryegrass.

Key words: CRISPR/Cas9, tRNA, multiple gene editing, vector, plasmid, protoplasts, perennial ryegrass