Multiplexed promoter and gene editing in wheat using the virus-based guide RNA delivery system

Abstract

The low efficiency of genetic transformation and gene editing across diverse cultivars hinders the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we applied the barley stripe mosaic virus (BSMV) for delivering guide RNAs (sgRNA) into the Cas9-expressing wheat lines to create targeted deletions in the promoter of a transcription factor and to perform multiplexed editing of agronomic genes. We demonstrate that pooled BSMV-sgRNAs could be used to generate heritable targeted deletions and multiple mutations in the genome. We transferred the high-expressing allele of Cas9 into adapted spring and winter cultivars and successfully performed the BSMV-sgRNA-based editing of two agronomic genes. The strategies presented in our study could be applied to any cultivar for creating new cis-regulatory diversity or targeting multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes and accelerating gene discovery and trait improvement efforts.