Crispr-cas9 editing of psbs uorfs to enhance intrinsic water use efficiency in cowpea

Overview

This project applies CRISPR-Cas9 gene editing to increase the intrinsic water use efficiency (iWUE) of cowpea, a vital legume crop for food security in sub-Saharan Africa. Rather than introducing foreign genes, the approach makes minimal edits to the cowpea genome by disrupting repressive upstream open reading frames (uORFs) in the transcript leader of the photosystem II subunit S (PsbS) gene. Prior work showed that overexpressing PsbS improves iWUE in tobacco, and separate studies demonstrated that removing repressive uORFs can increase expression of the target gene. By combining these insights, the technology aims to deliver drought-tolerant cowpea varieties with only minor, precisely targeted changes to the plant's DNA.

Technical specifications

• Target gene: PsbS (photosystem II subunit S), associated with improved intrinsic water use efficiency and drought tolerance
• Editing method: CRISPR-Cas9 disruption of two identified repressive uORFs in the cowpea PsbS transcript leader
• Validation assay: A dual-fluorescence screen in Nicotiana benthamiana confirmed that disruption of the identified uORFs increased reporter signal by over 100 percent (n = 8)
• Cross-species support: Comparable repressive uORFs were identified in soybean PSBS transcript leaders, reinforcing the biological relevance of the target
• Editing workflow: Three to five CRISPR constructs will be designed and cloned, followed by transient expression testing in cowpea leaf tissue to evaluate sgRNA targeting efficiency
• Stable line development: Stable cowpea mutant lines will be generated through project collaborators, with Cas9 null segregants potentially available as early as the T1 generation

Technology readiness level

The technology is at an early-to-mid stage of development. The uORF targets have been identified and validated using a robust dual-fluorescence assay, and CRISPR construct design and cloning are the next steps, with an estimated timeline of approximately three months. Transient screening in cowpea is underway through an ongoing collaboration. Stable mutant lines and Cas9-free segregants are anticipated by the end of 2023. Further field-level validation of iWUE gains and agronomic performance will be needed before commercial deployment.