An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design

Sci Rep. 2016 Apr 1:6:23890. doi: 10.1038/srep23890.

Abstract

Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis Proteins / genetics
  • CRISPR-Cas Systems*
  • Gene Deletion
  • Gene Editing / methods*
  • Gene Knockout Techniques
  • Genes, Plant*
  • Genetic Vectors / genetics*
  • MicroRNAs / genetics
  • Mutation
  • Plants, Genetically Modified
  • RNA, Plant / genetics
  • Transformation, Genetic

Substances

  • Arabidopsis Proteins
  • MIRN169 microRNA, Arabidopsis
  • MIRN827 microRNA, Arabidopsis
  • MicroRNAs
  • RNA, Plant
  • TFL1 protein, Arabidopsis