Crispr/cas9 genome editing platform for cassava shelf life improvement

Overview

This solution applies CRISPR/Cas9 genome editing to cassava, one of Africa's most important staple crops, to address its notoriously short post-harvest shelf life. By targeting the Feruloyl CoA 6'-Hydroxylase gene, the approach reduces scopoletin accumulation, a key contributor to rapid post-harvest deterioration. The platform uses cassava protoplasts as explants and delivers CRISPR ribonucleoproteins (RNPs) via polyethylene glycol (PEG), enabling targeted mutagenesis in a non-GMO manner. This makes the technology attractive for regions with restrictive GMO regulations and for markets demanding non-transgenic crop improvement.

The work is grounded in prior validation showing successful protoplast isolation and regeneration across three cassava cultivars (TMS60444, Muchericheri, and Karibuni), with the local Kenyan variety Muchericheri outperforming the model cultivar TMS60444 in regenerability. The platform offers cassava breeders, seed companies, and agricultural development programs a pathway to improved varieties with longer shelf life, reduced post-harvest losses, and enhanced food security.

Technical specifications

Key features:

• CRISPR/Cas9 ribonucleoprotein delivery into cassava mesophyll-derived protoplasts using PEG-mediated transformation
• Targeted mutagenesis of the Feruloyl CoA 6'-Hydroxylase gene to reduce scopoletin biosynthesis
• Validated protoplast regeneration system spanning solid and suspension culture, colony formation, cotyledonary embryo development, and germination on hormone-free MS medium with 3% sucrose
• Successful transfer of regenerated plants to greenhouse with development into fertile, mature plants
• Non-GMO approach using RNP delivery rather than plasmid DNA integration
• Demonstrated across multiple cultivars including TMS60444, Muchericheri, and Karibuni, with Muchericheri showing superior protoplast regenerability

Technology readiness level

The protoplast isolation and regeneration pipeline has been validated through prior experimental work, with successful culture initiation, embryo formation, plantlet germination, and greenhouse establishment of fertile plants. The CRISPR/Cas9 mutagenesis step, targeting Feruloyl CoA 6'-Hydroxylase to reduce scopoletin and extend shelf life, is the next validation phase and is estimated to take approximately one year to complete. The current readiness level is early-to-mid stage development (TRL 3-4), with biological proof-of-concept established and the genome editing application pending validation in transformed protoplasts.