TY - JOUR T1 - Optimizing multiplex CRISPR/Cas9-based genome editing for wheat JF - bioRxiv DO - 10.1101/051342 SP - 051342 AU - Wei Wang AU - Alina Akhunova AU - Shiaoman Chao AU - Eduard Akhunov Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/05/04/051342.abstract N2 - Background CRISPR/Cas9-based genome editing holds great promise to accelerate the development of improved crop varieties by providing a powerful tool to modify the genomic regions controlling major agronomic traits. The effective deployment of this technology for crop improvement still requires further optimization of the components of the CRISPR/Cas9 system for different applications. Here we have optimized a multiplex gene editing strategy for hexaploid wheat.Results The functionality of the various components of the CRISPR/Cas9 gene editing system was validated using the wheat protoplast transformation assay followed by the next-generation sequencing (NGS) of the targeted genomic regions. The wheat codon optimized Cas9 was shown to be effective tool for the targeted gene editing in the wheat genome. Multiple single guide RNAs (gRNAs) were evaluated for the ability to target the homoeologous copies of four genes controlling important agronomic traits in wheat. Low correspondence was found between the gRNA efficiency predicted bioinformatically and that assessed in the protoplast transient expression assay. Multiplex gene editing was successfully accomplished using the construct with several gRNA-tRNA units under the control of a single promoter for the RNA polymerase III.Conclusions Here we demonstrate that by integrating the fast transient expression assay in the protoplasts with multiplexed NGS, it is possible to perform functional screens for a large number of gRNAs and to optimize constructs for effective editing of multiple independent targets in the wheat genome. We further showed that multiplex gene editing in wheat can be effectively accomplished using a construct with multiple tandemly arrayed tRNA–gRNA units under the control of the wheat U3 snoRNA promoter. A polycistronic gene construct that can be quickly assembled using the Golden Gate reaction along with the wheat codon optimized Cas9 further expand the set of tools available for engineering the wheat genome. ER -