Creating targeted gene knockouts in barley using CRISPR/Cas9

Abstract

Knockout mutants are an invaluable reverse genetics tool which have not been well developed in crop species compared to models like Arabidopsis. However, the emergence of CRISPR/Cas9 has changed this situation making the generation of such mutants accessible to many crops including barley. A single T-DNA construct can be transformed into barley immature embryos and stable transgenic lines regenerated through tissue culture which contain targeted mutations. Mutations are detected in T0 plants and go on in subsequent T1 and T2 generations to segregate from T-DNA, leaving lines which are non-transgenic and carrying a variety of mutations at the target locus. These mutations can be targeted to a particular gene of interest in order to bring about a loss of function creating a knockout mutant.