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

Zhang, Zhengjing, Mao, Yanfei, Ha, Si, Liu, Wenshan, Botella, Jose Ramon and Zhu, Jian-Kang (2015) A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis. Plant Cell Reports, 1-15. doi:10.1007/s00299-015-1900-z

Author Zhang, Zhengjing
Mao, Yanfei
Ha, Si
Liu, Wenshan
Botella, Jose Ramon
Zhu, Jian-Kang
Title A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis
Journal name Plant Cell Reports   Check publisher's open access policy
ISSN 0721-7714
Publication date 2015-12-10
Year available 2015
Sub-type Article (original research)
DOI 10.1007/s00299-015-1900-z
Open Access Status Not yet assessed
Start page 1
End page 15
Total pages 15
Place of publication Heidelberg, Germany
Publisher Springer
Collection year 2016
Language eng
Formatted abstract
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.
Keyword CRISPR/Cas9
Gene editing
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2016 Collection
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