Wheat genome editing through wide hybridization with maize expressing Cas9 and guide RNA

Authors: KARMACHARYA, ANIL - North Dakota State University; Li, Dandan; LENG, YUEQIANG - North Dakota State University; SHI, GONGJUN - North Dakota State University; LIU, ZHAOHUI - North Dakota State University; Yang, Shengming; DU, YANG - Valley City State University; DAI, WENHAO - North Dakota State University; ZHONG, SHAOBIN - North Dakota State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/5/2023
Publication Date: 12/7/2023
Citation: Karmacharya, A., Li, D., Leng, Y., Shi, G., Liu, Z., Yang, S., Du, Y., Dai, W., Zhong, S. 2022. Wheat genome editing through wide hybridization with maize expressing Cas9 and guide RNA. Meeting abstract. 2022 National FHB Forum. Poster No. 139.

Interpretive Summary:

Technical Abstract: CRISPR/Cas9-mediated genome editing technology is a powerful tool for targeting genes of interest in various crops. In wheat, most of the genome editing studies so far have directly or indirectly relied on callus induction and plant regeneration from the explants receiving gene construct or preassembled Cas9/gRNA ribonucleoprotein complex delivered by particle bombardment or Agrobacterium-mediated methods. Unfortunately, the efficiency of callus induction and plant regeneration is generally very low for most wheat genotypes, limiting the application of genome editing in many commercially grown wheat varieties. Recently, a new genome editing approach named “Haploid Inducer-Mediated Genome Editing” or “HI-Edit” has been used to generate transgene-free doubled haploid plants with the target genes edited. However, few studies have been reported on targeting wheat genes using this approach. In this study, we aimed to use HI-edit to target two genes, TaHRC and Tsn1, which are involved in susceptibility to Fusarium head blight (FHB) and three foliar diseases (tan spot, Septoria nodorum blotch and spot blotch), respectively, in wheat. For each gene, two target sites were selected to make a gene construct that contains the components of CRISPR/Cas9-mediated genome editing machinery for wheat. The gene constructs were used to transform the hybrid maize variety Hi-II by Agrobacterium-mediated transformation. The resulting T0 and T1 maize plants with high expression of Cas9 and gRNA were used to pollinate emasculated spikes of wheat variety Dayn. Haploid wheat plants were generated from the tissue-culture rescued embryos derived from the wide crosses. PCR amplification and sequencing indicated that 15-33% of the haploid plants contained mutations at the target sites of the targeted gene. Doubled haploid plants with mutations at the target genes were generated and phenotype data on disease