Accepted_test

Barley is a key cereal crop in agriculture and the food industry. This study aimed to use targeted genome editing to knock out genes involved in fatty acid metabolism, specifically the cytochrome P450 gene and three genes encoding ketoacyl-CoA synthetases. Transcriptomic analysis of barley mutants for the HvWin1 gene, involved in wax biosynthesis, identified these four genes with significantly altered expression.

CRISPR/Cas9 vector constructs were developed using gene sequences from the Golden Promise barley cultivar. Benchling and WU-CRISPR tools identified optimal cleavage sites for guide RNAs (gRNAs), and their secondary structure was predicted using RNAfold. The expression vectors included Cas9 nuclease coding sequence, gRNA, and the U6 promoter from Triticum aestivum. These constructs, designated as pEK7, pEK13, pEK17, and pEK25, were cloned into a binary vector and used to transform immature Golden Promise barley embryos via Agrobacterium-mediated transformation.

This transformation resulted in transgenic barley lines with mutations in the target genes: 23 plants for the cytochrome P450 gene and 38, 44, and 23 plants for the three ketoacyl-CoA synthetase genes. These lines will be analyzed in the T1 generation for changes in fatty acid composition compared to wild-type plants.

The study successfully applied a CRISPR/Cas9 system for genome editing in barley, creating transgenic lines with targeted gene knockouts. This research provides a basis for further exploration of fatty acid biosynthesis in barley, aiming to improve crop quality and agricultural productivity. The findings have significant implications for developing new barley cultivars with enhanced traits, potentially boosting agricultural efficiency.