Plant Cell Reports

, Volume 35, Issue 7, pp 1519–1533 | Cite as

A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis

  • Zhengjing Zhang
  • Yanfei Mao
  • Si Ha
  • Wenshan Liu
  • Jose Ramon Botella
  • Jian-Kang ZhuEmail author
Original Article


The recently developed CRISPR/Cas9 system is a promising technology for targeted genome editing in a variety of species including plants. However, the first generation systems were designed to target one or two gene loci at a time. We designed a new multiplex CRISPR/Cas9 system that allows the co-expression of six sgRNA modules in one binary vector using a simple (three steps) cloning strategy in Arabidopsis. The transcription of the sgRNA modules is under the control of three different RNA Polymerase III-dependent promoters. We tested the efficiency of the new multiplex system by targeting six of the fourteen PYL families of ABA receptor genes in a single transformation experiment. One line with mutations in all six targeted PYLs was identified from 15 T1 plants. The mutagenesis frequency for the six individual PYL targets in the T1 lines ranged from 13 to 93 %. In the presence of ABA, the transgenic line identified as containing mutations in all six PYL genes produced the highest germination rate in the T2 progeny (37 %). Among these germinated seedlings, half of the analyzed plants (15/30) were homozygous mutants for at least four targeted genes and two plants (6.7 %) contained homozygous mutations in five of the targeted PYLs and the other targeted PYL had biallelic mutations. Homozygous sextuple mutants were identified in the T3 progeny and characterized together with previously described triple and sextuple PYL mutants. We anticipate that the application of this multiplex CRISPR/Cas9 system will strongly facilitate functional analysis of genes pathways and families.


CRISPR/Cas9 Multiplex Gene editing Arabidopsis 



We thank Dr Pedro L. Rodriguez for kindly providing the pyr1pyl1pyl4 and pyr1pyl1pyl2pyl4pyl5pyl8 mutants. The work was supported by the Chinese Academy of Sciences. The authors declare that they have no conflicts of interest with respect to this work.

Supplementary material

299_2015_1900_MOESM1_ESM.pdf (343 kb)
Supplementary material 1 (PDF 343 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Zhengjing Zhang
    • 1
    • 2
  • Yanfei Mao
    • 1
  • Si Ha
    • 1
    • 2
  • Wenshan Liu
    • 1
    • 5
  • Jose Ramon Botella
    • 3
  • Jian-Kang Zhu
    • 1
    • 4
    Email author
  1. 1.Shanghai Center for Plant Stress Biology, Chinese Academy of SciencesShanghaiChina
  2. 2.University of Chinese Academy of Sciences (CAS)ShanghaiPeople’s Republic of China
  3. 3.School of Agriculture and Food SciencesUniversity of QueenslandBrisbaneAustralia
  4. 4.Department of Horticulture and Landscape ArchitecturePurdue UniversityWest LafayetteUSA
  5. 5.School of Life SciencesChongqing UniversityChongqingChina

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