Using CRISPR/Cas9 System to Introduce Targeted Mutation in Arabidopsis

Methods Mol Biol. 2018:1830:93-108. doi: 10.1007/978-1-4939-8657-6_6.

Abstract

Gene editing enables the modification of genetic information in a targeted manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing approach that has been widely used in both the animal and plant studies. Plant transcription factors play critical roles in plant development. Recent studies have used the CRISPER/Cas9 system to disrupt transcription factors and cis-elements in plants. In this chapter, we describe a protocol of using the CRISPR/Cas9 system to generate transcription factor mutants in the model plant species Arabidopsis thaliana. Our protocol contains five different steps: (1) adaptor primers design, (2) cloning of sgRNA expression cassettes, (3) Golden Gate cloning, (4) E. coli and Agrobacterium transformation, and (5) Arabidopsis transformation.

Keywords: Arabidopsis; CRISPR/Cas9; DNA-binding domain; Gene editing; Plant transcription factors; cis-elements.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium / genetics
  • Arabidopsis / genetics*
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • DNA Primers / metabolism
  • Genome, Plant
  • Molecular Biology / methods*
  • Mutagenesis / genetics
  • Mutation / genetics*
  • Physical Chromosome Mapping
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Transformation, Genetic

Substances

  • DNA Primers
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9