Crispr-cas9 soybean lines with high oleic acid and dual herbicide tolerance

Overview

This solution delivers gene-edited soybean lines that simultaneously address two critical challenges for soybean producers and food manufacturers: oil quality and weed management. By editing the native FAD2 gene and its isoforms, the soybean produces oil with a higher oleic acid (Omega 9, C18:1) to linoleic acid ratio, delivering health benefits and enhanced oil stability, flavor, and nutrition. Concurrently, the introduction of three mutations in the PEP binding domain of the native EPSPS gene and two mutations in the native ALS gene confers tolerance to both glyphosate and sulfonylurea herbicides, reducing yield losses from weed infestation.

Oils rich in monounsaturated fatty acids such as oleic acid offer cardiovascular benefits and oxidative stability, making them suitable for diverse food and industrial applications. Dual herbicide tolerance enables flexible and effective weed control strategies, particularly valuable in intensive soybean cultivation systems.

Technical specifications

Key features:

• CRISPR-Cas9 and homology-directed repair (HDR) strategy applied to soybean
• Knockout of native FAD2 gene and its isoforms to elevate oleic acid content
• Three mutations introduced in the PEP binding domain of the native EPSPS gene for glyphosate tolerance
• Two mutations introduced in the native ALS gene for sulfonylurea tolerance
• Multiplexed expression cassettes combining pegRNAs (EPSPS + ALS + FAD2), HDR templates, and codon-optimized Cas9 + RT genes assembled into a prime editing binary vector
• Transformation via biolistic or agro-mediated approaches
• In-house designed vectors with demonstrated efficacy in maize, rice, and pigeon pea crops
• Established protocol for callus regeneration and transformation of Cas9 gene in soybean

Technology readiness level

The underlying CRISPR-Cas9 and HDR editing platform has been validated in multiple crops. The team has successfully introduced mutations in native EPSPS and ALS genes of maize, rice, and pigeon pea, and has generated glyphosate-tolerant maize lines with three mutations in the native ZmEPSPS gene, for which a patent application has been filed. Callus regeneration and transformation protocols for soybean have been developed. Next steps include designing pegRNAs and HDR templates using in silico tools, assembling multiplexed expression cassettes, transforming soybean varieties, and conducting molecular analyses and event characterization for high oleic acid content and dual herbicide tolerance.