Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

PLoS One. 2019 Jan 9;14(1):e0204778. doi: 10.1371/journal.pone.0204778. eCollection 2019.

Abstract

Bacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics
  • Arabidopsis / genetics*
  • CRISPR-Cas Systems / genetics*
  • DNA, Bacterial / genetics*
  • Gene Editing / methods*
  • Genetic Vectors
  • Mutagenesis, Site-Directed / methods*
  • Plants, Genetically Modified
  • Plasmids / genetics
  • Promoter Regions, Genetic / genetics
  • RNA, Guide, CRISPR-Cas Systems / chemistry
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Transformation, Bacterial / genetics

Substances

  • DNA, Bacterial
  • RNA, Guide, CRISPR-Cas Systems
  • T-DNA