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

Date Published: January 9, 2019

Publisher: Public Library of Science

Author(s): Baptiste Castel, Laurence Tomlinson, Federica Locci, Ying Yang, Jonathan D. G. Jones, Erh-Min Lai.


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.

Partial Text

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR associated) site-specific nucleases evolved as components of prokaryotic immunity against viruses, and are widely deployed as tools to impose operator-specified nucleotide sequence changes in genomes of interest [1–4]. During infection by bacteriophages, Cas1 and Cas2 can integrate phage DNA sequences into ‘spacer’ regions of tandem CRISPR loci in the bacterial genome. The crRNA (CRISPR-RNA) transcription product of the spacer associates with nucleases from the Cas family to form ribonucleoproteins that can cleave nucleic acid sequences homologous to the spacer. This enables elimination of viral nucleic acid upon subsequent infection. CRISPR systems are divided in two classes [5,6]. Class 1 systems comprise multi-subunit complexes whereas Class 2 systems function with single ribonucleoproteins. Within Class 2, Type-II and Type-V cleave dsDNA (double-stranded DNA) via Cas9 and Cas12/Cpf1 respectively, while Type-VI cleaves ssRNA (single-stranded RNA) via Cas13/C2c2.

CRISPR emerged in 2012 as a useful tool for targeted mutagenesis in many organisms including plants [11,32]. In Arabidopsis, the transgenic expression of CRISPR components can be straightforward, avoiding tedious tissue culture steps. Many strategies to enhance the overall CRISPR-induced mutation rate have been proposed [8,13,15–17,40]. Here we report a systematic comparison of putative limiting factors including promoters, terminators, codon optimization, sgRNA improvement and T-DNA architecture.




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