Knockout of the Bread Wheat CER9/SUD1 Gene Using CRISPR/Cas Technology

Abstract

Mutations in the CER9 gene of Arabidopsis thaliana L. contribute to the amplification of the cuticular wax and consequently mitigation of water loss, thereby fortifying drought resilience. Recently, genes homologous to CER9, termed SUD1 genes, have been annotated in bread wheat (Triticum aestivum L.). However, no research has been done on these genes in T. aestivum. Hence, our study aimed to employ CRISPR/Cas technology to knock out the CER9/SUD1 gene orthologs in bread wheat. For this, five guide RNAs were meticulously chosen and merged into a singular vector. Delivery of the CRISPR/Cas components was arranged through Agrobacterium tumefaciens, utilized for transforming immature embryos of two agricultural spring bread wheat varieties: Taya and Sigma. Among the 13 transgenic plants procured, four manifested positivity for the reporter gene GFP and Cas9 gene. Notably, substantial deletions ranging from 284 to 398 bp within the CER9/SUD1 gene were discerned in these plants. Additionally, two of the edited plants exhibited an absence of CER9/SUD1 transcripts, while the other two displayed a noteworthy 5.4-fold reduction in CER9/SUD1 gene expression compared to the wild type. Intriguingly, the genome-edited plants of the T₁ generation showcased enhanced growth parameters compared to the wild type under both standard and drought conditions.