title: A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis.
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.
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 200032, China.|University of Chinese Academy of Sciences (CAS), Shanghai, 200032, People's Republic of China.
authors.forename: Zhengjing
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 200032, China.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Mao
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 200032, China.|University of Chinese Academy of Sciences (CAS), Shanghai, 200032, People's Republic of China.
authors.forename: Si
authors.initials: S
authors.lastname: Ha
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 200032, China.|School of Life Sciences, Chongqing University, Chongqing, 400044, China.
authors.forename: Wenshan
authors.initials: W
authors.lastname: Liu
authors.affiliation: School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, 4072, Australia.
authors.forename: Jose Ramon
authors.initials: JR
authors.lastname: Botella
authors.affiliation: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, Shanghai, 200032, China. jkzhu@purdue.edu.|Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, 47907, USA. jkzhu@purdue.edu.
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics*; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed / Q000379:methods; D030821:Plants, Genetically Modified
chemical_list: D029681:Arabidopsis Proteins
keywords: Arabidopsis; CRISPR/Cas9; Gene editing; Multiplex