Abstract
BackgroundThe 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.ResultsWe 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.ConclusionOur 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.
Highlights
The use of Clustered regularly interspaced short palindromic repeats (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
The system requires two components to be expressed within the same plant cell, the Cas9 adapted from Streptococcus pyogenes and a short guide RNA which targets the Cas9 to the required genomic sequence
Sufficient differences were identified within the introns of The wheat phytoene desaturase (TaPDS) homoeologues to enable design of genome specific primers for PCR amplification (Additional file 2: Table S1), while exonic similarity was sufficient to allow guide RNA (gRNA) sequences to be identified for both genome-specific edits and to target all three homoeologues simultaneously with a guide of 100% match (Additional file 1: Figure S1)
Summary
The use of CRISPR/Cas 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. The ability to examine valuable agronomic traits within crop species has, until recently, been possible only by extensive crossing programmes or the use of genetic modification to insert or silence target genes. Recent developments in the area of gene editing could, be invaluable as a means of reliably producing true knockouts, in polyploid species. The use of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems is increasingly reported in plant species. Despite the progress made in plants, gene editing remains challenging in transformation-recalcitrant species, with relatively few experiments reported in the major crop species. Within the monocots, editing has been reported in maize, rice and barley [2,3,4]; questions remain from these studies regarding both the editing efficiency and heritability of the induced edits [5, 6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
