Crispr-cas9 gene editing to boost beta-carotene content and stress tolerance in sweet potatoes

Overview

Vitamin A deficiency remains a significant public health challenge in many developing countries, contributing to preventable blindness, weakened immunity, and increased child mortality. Sweet potato is a widely consumed staple crop that can serve as a vehicle for delivering pro-vitamin A (beta-carotene) to vulnerable populations. This project applies CRISPR-Cas9 gene editing to knock out the beta-carotene hydroxylase gene, which is responsible for converting beta-carotene into other compounds. By reducing the activity of this gene, beta-carotene accumulates in the sweet potato tissues, increasing its nutritional value. The approach also leverages the observed association between high beta-carotene content and improved tolerance to environmental stresses such as drought and salinity, offering dual benefits for food security and crop resilience.

Technical specifications

• Target gene: Beta-carotene hydroxylase, edited using CRISPR-Cas9 to reduce its activity and promote beta-carotene accumulation
• Editing method: CRISPR-Cas9 mediated genome editing delivered via Agrobacterium-mediated transformation
• Construct design: Single guide RNAs (sgRNAs) synthesized and assembled into a binary vector for transformation
• Transformation protocol: Optimized regeneration and transformation protocol with 3-8% regeneration efficiency in selected sweet potato genotypes, developed through prior PhD research
• Variety selection: Four sweet potato varieties chosen based on consumer preference, transformability, and cross-pollination ability
• Transgene removal: Final-stage cross-pollination strategy to remove transgenes from selected edited lines, producing transgene-free improved varieties
• Prior validation: RNA interference knockdown of the same gene previously demonstrated increased beta-carotene content and improved stress tolerance in transgenic sweet potato lines

Technology readiness level

The project is at an early-to-mid stage of development. Prior RNA interference work has provided proof-of-concept that downregulating the beta-carotene hydroxylase gene increases beta-carotene content and stress tolerance. CRISPR-Cas9 editing has already been demonstrated in sweet potatoes, and transformation and regeneration protocols have been optimized with measurable efficiency improvements. The remaining validation involves a four-stage pipeline: sequencing the target gene across selected varieties, constructing editing vectors, transforming and regenerating edited lines, and removing transgenes through cross-pollination. The technology is not yet commercially deployed and requires completion of gene editing, transgene removal, and field validation before reaching market readiness.