Application of Multiplex CRISPR/Cas9-Based Genome Editing Strategy for Targeting Multiple Agronomic Genes in Wheat.

The CRISPR/Cas9-based genome editing tools hold a great promise to accelerate crop breeding by providing means to modify genes controlling important agronomic traits. We have established the CAS9-based gene editing pipeline for wheat that includes the following components: wheat codon optimized CAS9 construct; procedures for the high-throughput screening of the designed gRNAs for their editing efficiency using the wheat protoplast assay; procedures for the next-generation sequencing of multiplexed amplicons generated for the targeted genomic regions; bioinformatical pipeline for the quick assessment of the frequency and types of editing events in the wheat genome; simplex and multiplex (tRNA-based spacer separated) gene editing constructs (MGEC) that can be assembled using the Golden Gate strategy.

The efficiency of the established gene editing pipeline was demonstrated by modifying four genes controlling agronomic traits in wheat. In protoplasts, a MGEC with several gRNA-tRNA units under the control of a single promoter was shown to generate indels at the targets sites with the efficiency comparable to that obtained for single gRNA constructs. Editing events in multiple genes were detected in the wheat plants regenerated from the immature embryos transformed with the MGEC. The phenotypic evaluation of plants carrying mutations on all homoeologues copies of the TaGW2 gene induced by the MGEC showed increase in the thousand grain weight, grain area, grain width, and grain length, which was almost two times higher than that observed in the plants carrying mutations in only one or two homoeologues copies of the TaGW2 gene. Here, we demonstrate that the multiplexing capacity of the tandemly arrayed tRNA–gRNA constructs is well suited for the simultaneous editing of the redundant gene copies in the allopolyploid genomes or genomic regions beneficially affecting multiple agronomic traits. The developed resources further expand the set of tools available for engineering the wheat genome.