Optimizing multiplex CRISPR/Cas9-based genome editing for wheat

Background: CRISPR/Cas9-based genome editing holds a great promise to accelerate the development of improved crop varieties by providing a powerful tool to modify genomic regions controlling major agronomic traits. The effective deployment of this technology for crop improvement still requires further optimization of the CRISPR/Cas9 system for different applications. Here we have optimized 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 next-generation sequencing (NGS) of the targeted gene regions. The wheat codon optimized Cas9 was shown to be able to effectively edit targets in the wheat genome. Multiple 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 RNA polymerase III promoter. Conclusions: The assessment of gRNAs’ targeting efficiency using the transient expression in the protoplasts and NGS suggest that further optimization of the gRNA design tools will be required to improve the selection of functional gRNAs for the allopolyploid genome. The empirically selected gRNAs provided an effective method for designing a successful multiplex gene editing experiment in wheat. Wheat codon optimized Cas9 as well as the Golden Gate-compatible tRNA processing-based gRNA cassette developed here further expand the CRISPR/Cas9 toolbox available for editing the hexaploid wheat genome. not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/051342 doi: bioRxiv preprint first posted online May. 2, 2016;