Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions in Barley and Generation of Homozygous Edited Lines via Embryogenic Pollen Culture

Methods Mol Biol. 2018:1789:9-20. doi: 10.1007/978-1-4939-7856-4_2.

Abstract

The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.

Keywords: CRISPR/Cas9; Cereals; Doubled haploid; GC rich sequence; Genome editing; Hordeum vulgare cv. “Golden Promise”; Induced mutation frequency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • DNA, Plant / genetics
  • GC Rich Sequence
  • Gene Deletion
  • Gene Editing / methods*
  • Genome, Plant
  • Haploidy
  • Hordeum / embryology
  • Hordeum / genetics*
  • Mutation Rate
  • Pollen / embryology
  • Pollen / genetics*
  • Tissue Culture Techniques / methods
  • Transformation, Genetic

Substances

  • DNA, Plant