Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9

Abstract

Background: The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles.

Results: We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11-17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing.

Conclusion: Our work shows the potential to obtain stable edited transgene-free wheat lines in 36 weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies.

Keywords: CRISPR\Cas9; Genome editing; Heritability; Hordeum vulgare; Knockout; Triticum aestivum.

Fig. 1

Schematic of binary plasmid T-DNA region transferred to wheat or barley. All constructs conform to this structure with differences as indicated in Table 1 for the specific Pol III promoters included

Fig. 2

Transgenic barley plant exhibiting chimaeric photobleaching phenotype