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. To diversify wheat CRISPR/Cas9 toolbox, we have developed a wheat codon-optimized Cas9 and tested the performance of the tRNA-based multiplex gene editing constructs (MGEC) in hexaploid wheat. We showed that the efficiency of the CRISPR/Cas9 editing can be effectively assessed using the transient expression in the wheat protoplasts followed by the next-generation sequencing (NGS) of the targeted genomic regions. The ability of gRNAs to perform homoeolog-specific target editing as well as simultaneous editing of multiple homoeologs was demonstrated by modifying four genes known to affect agronomic traits in wheat. In protoplasts, a multiplex gene editing construct 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 wheat plants regenerated from the immature embryos transformed with the multiplex editing construct. 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. These gene constructs can be quickly assembled using the Golden Gate reaction and along with the wheat codon-optimized Cas9 further expand the set of tools available for engineering the wheat genome.

 Keywords

CRISPR/Cas9, genome editing, polyploid wheat, multiplex editing, tRNA