title: A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
abstract: BACKGROUND
  To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.
  
  
  RESULTS
  We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.
  
  
  CONCLUSIONS
  We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
authors.forename: Hui-Li
authors.initials: HL
authors.lastname: Xing
authors.forename: Li
authors.initials: L
authors.lastname: Dong
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Wang
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Zhang
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Han
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Xue-Chen
authors.initials: XC
authors.lastname: Wang
authors.forename: Qi-Jun
authors.initials: QJ
authors.lastname: Chen
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant; D030821:Plants, Genetically Modified / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism; D016415:Sequence Alignment; D003313:Zea mays / Q000235:genetics*