Here we present a rapid, inexpensive screening method for edited genes in transgenic wheat plants. Sequencing of homoeolog-specific amplicons is most often used for this.
Identifying specific editing events in transgenic wheat plants is challenging due to the high homology of genes among the three subgenomes and the number of duplication events for those genes.
The utility of paired sgRNA-based approach for targeted editing of homoeologous genes has been tested in transgenic wheat plants for TaNFXL1. In addition, a direct comparison of editing efficiency in wheat protoplasts was performed with two modified nucleases Cas9, crCas9 and pcoCas9, both derived from the Streptococcus pyogenes Cas9. These three genes were previously identified to be associated with susceptibility ( TaABCC6, TaNFXL1) and resistance ( TansLTP9.4) to Fusarium head blight (FHB), a devastating fungal disease of wheat. Here, we explore this approach, and present a protocol for targeted deletion of gene fragments using paired sgRNA and the CRISPR/Cas9 system in wheat and validate it for three wheat genes in a wheat protoplast system: an ABC transporter ( TaABCC6), a lipid transfer protein ( TansLTP9.4) and a putative transcription repressor named TaNFXL1. However loss-of-function mutants with larger deletion, preferably in most of the homoeologs for a given gene, are desired to observe a phenotypic change in wheat., The use of paired sgRNA for CRISPR/Cas9 editing of a targeted gene has been shown to result in larger deletions in Arabidopsis thaliana, rice and kiwifruit however this method has not been tested in wheat. Įditing events obtained in wheat using CRISPR/Cas9 and one single guide RNA (sgRNA) are often associated with single nucleotide deletion/addition or small deletions. The size of the wheat genome is approximately 16 Gb over 80% of it is composed of highly repetitive sequences and transposable elements and estimated to encode 108,000 high-confidence protein-coding loci. The wheat genome comprises three subgenomes (A, B and D), each representing a set of seven chromosomes. īread wheat ( Triticum aestivum) plays a central role in global food and feed crop consumption and is one of the most widely cultivated crops around the world. Also, this technology has been successfully applied for gene editing in several crops including rice ( Oryza sativa), maize ( Zea mays) and sorghum ( Sorghum bicolor) genomes. To date, several genes targeted in model plants such as Arabidopsis thaliana and tobacco ( Nicotiana tabacum) have been successfully edited using this technology. In plants, this technology has been shown to have a high potential for gene editing both in monocot and dicot plants. The first application of CRISPR as a gene editing tool was reported in human and mouse cells, demonstrating that Cas9 nucleases could induce precise cleavage at targeted genome loci with the presence of short RNA guiding sequences.
The type II prokaryotic Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system was initially identified in 2007, and the unique features of this system have been explored widely in both Eubacteria and Archaea.