Journal Article

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

Jones, Jonathan D. G., Castel, Baptiste, Yang, Ying, Tomlinson, Laurence, Locci, Federica

2019

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Agrobacterium tumefaciens - genetics / Alcohol / Alleles / Analysis / Arabidopsis / Arabidopsis - genetics / Architecture / Biology and Life Sciences / Cloning / CRISPR / CRISPR-Cas systems / CRISPR-Cas Systems - genetics / Deoxyribonucleic acid / DNA / DNA, Bacterial - genetics / Efficiency / Engineering and Technology / Functional analysis / Gene Editing - methods / Gene expression / Gene families / Gene loci / Genetic aspects / Genetic modification / Genetic transformation / Genetic Vectors / Genome editing / Genomes / Laboratories / Methods / Mutagenesis / Mutagenesis, Site-Directed - methods / Mutation / Nuclease / Optimization / Plants, Genetically Modified / Plasmids - genetics / Promoter Regions, Genetic - genetics / Research and Analysis Methods / RNA polymerase / RNA, Guide, CRISPR-Cas Systems - chemistry / RNA, Guide, CRISPR-Cas Systems - genetics / T-DNA / Transcription / Transformation / Transformation, Bacterial - genetics