Optimizing multiplex CRISPR/Cas9-based genome editing for wheat
BackgroundCRISPR/Cas9-based genome editing holds great promise to accelerate the development of new crop varieties by providing a powerful tool to modify the genomic regions controlling major agronomic traits. To diversify the set of tools available for wheat genome engineering, we have established a tRNA-based multiplex gene editing strategy for hexaploid wheat.\n\nResultsThe functionality of the various CRISPR/Cas9 components was assessed using the transient expression in the wheat protoplasts followed by next-generation sequencing (NGS) of the targeted genomic regions. The efficiency of wheat codon-optimized Cas9 for targeted gene editing in wheat was validated. Multiple single guide RNAs (gRNAs) were evaluated for the ability to edit the homoeologous copies of four genes affecting some important agronomic traits in wheat. Low correspondence was found between the gRNA efficiency predicted bioinformatically and that assessed in the transient expression assay. A multiplex gene editing construct with several gRNA-tRNA units under the control of a single promoter for the RNA polymerase III generated indels at the targets sites with the efficiency comparable to that obtained for a single gRNA construct.\n\nConclusionsBy integrating the protoplast transformation assay with multiplexed NGS, it is possible to perform fast functional screens for a large number of gRNAs and to optimize constructs for effective editing of multiple independent targets in the wheat genome. The multiplexing capacity of the tandemly arrayed tRNA-gRNA construct is well suited for the simultaneous editing of the redundant gene copies in the allopolyploid genomes or genomic regions beneficially affecting multiple agronomic traits. A polycistronic gene construct that can be quickly assembled using the Golden Gate reaction along with the wheat codon optimized Cas9 will further expand the set of tools available for engineering the wheat genome.