CRISPR/Cas9 in a Complex and Polyploid Genome, Wheat.

The efficiency of CRISPR/Cas9 system has been shown in several studies using diploid plants, however, its application and stability still remain challenge for polyploid and complex genomes such as wheat. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of autophagy related genes Atg8 and Atg5 that we earlier identified and characterized in wheat and brachypodium. The targeted genome editing for these two genes was achieved and confirmed by using mutagenesis in wheat protoplast with restriction enzyme digestion assay, and T7 endonuclease assay. The off-target rate and specific copies of target genes from each sub-genome were analyzed and confirmed with amplicon sequencing. We also compare these results with immature embryo results. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast.