Virus-based guide RNA delivery facilitates multiplexed gene and promoter editing in wheat

Authors: WANG, WEI - Kansas State University; YU, ZITONG - Kansas State University; HE, FEI - Kansas State University; Bai, Guihua; TRICK, HAROLD - Kansas State University; AKHUNOVA, ALINA - Kansas State University; AKHUNOV, EDUARD - Kansas State University

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 4/6/2022
Publication Date: N/A
Citation: N/A
DOI: https://doi.org/10.1101/2022.04.06.484365

Interpretive Summary: Gene editing using CRISPR technologies is an emerging technique with broad applications to all of biological science. In wheat, the usefulness of gene editing has been hindered because only a few lines are easy to edit. To break this limitation on wheat gene editing, we used the barley stripe mosaic virus (BSMV) for delivering guide RNA to perform gene editing. We showed that the performance of the BSMV system strongly depended on the particular guide RNAs. We showed that multiple genes could be edited simultaneously, but the efficiency decreased as the number of targets increased. The strategies presented in our study open the possibilities for the effective engineering of the wheat genome and accelerating gene discovery and trait improvement efforts.

Technical Abstract: The low efficiency of genetic transformation and gene editing across diverse wheat cultivars hinders the broad application of CRISPR technologies for improving this crop. The development of virus-based guide RNA (sgRNA) delivery methods of CRISPR system into the plant cells holds great promise to overcome these limitations. Here, we applied the barley stripe mosaic virus (BSMV) for delivering sgRNA into the Cas9-expressing wheat lines to create deletions in the promoter of a transcription factor controlling domestication traits and to induce mutations in multiple genes. We show that the performance of the BSMV-sgRNA system strongly depends on the efficiency of sgRNAs and the levels of Cas9 expression. We show that even though multiplex editing could be achieved by inoculating plants with the pooled BSMV-sgRNA transcripts, the efficiency of individual target editing drops substantially with increase in the multiplexing level. We demonstrate that the targeted deletions of genomic regions within a gene promoter could be performed using the pooled BSMV-sgRNAs. We transferred the high-expressing allele of Cas9 into spring and winter cultivars and successfully performed editing of two agronomic genes by BSMV-sgRNAs inoculation. 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 the wheat genome and accelerating gene discovery and trait improvement efforts.