title: High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize.
abstract: CRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice.
  
  
  
  We optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize.
  
  
  
  The examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China.|Coordinated Crop Biology Research Center (CBRC), Beijing, 100193, China.
authors: Weiwei
authors: W
authors: Qi
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China.
authors: Tong
authors: T
authors: Zhu
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China.
authors: Zhongrui
authors: Z
authors: Tian
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China.
authors: Chaobin
authors: C
authors: Li
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China.
authors: Wei
authors: W
authors: Zhang
authors: Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 333 Nanchen Road, Shanghai, 200444, China. rentaosong@staff.shu.edu.cn.|National Maize Improvement Center of China, China Agricultural University, Beijing, 100193, China. rentaosong@staff.shu.edu.cn.|Coordinated Crop Biology Research Center (CBRC), Beijing, 100193, China. rentaosong@staff.shu.edu.cn.
authors: Rentao
authors: R
authors: Song
mesh_terms: D064130:CRISPR-Associated Proteins; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000072669:Gene Editing / Q000379:methods*; D017343:Genes, Plant / Q000235:genetics*; D005998:Glycine / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D003313:Zea mays / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D010940:Plant Proteins; D012343:RNA, Transfer; D005998:Glycine
keywords: CRISPR/Cas9; Maize; Multiplex gene editing; tRNA-processing