Developing a Molecular Toolkit to ENABLE all to apply CRISPR/Cas9-based Gene Editing in planta

Abstract

Gene editing, especially the CRISPR/Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system, has revolutionized trait development in crops. However, large parts of the world are missing out on applying CRISPR in planta. There is an obvious lack of gene editing applications in locally relevant crops in the Global South which tend to be neglected by mainstream agricultural research and development. Access barriers to these new breeding technologies need to be removed to allow the potential impact of these technologies on food security to happen. Here, we present the ENABLE(R) Gene Editing in planta toolkit, a minimal molecular toolbox allowing users to create a CRISPR knockout vector for transient or stable plant transformation in two simple cloning steps. We validate the toolkit in rice (Oryza sativa) protoplasts and in Arabidopsis thaliana plants. The ENABLE(R) kit is designed to be utilized specifically by users in the Global South who are new to CRISPR technology by providing a simple workflow, extensive accompanying protocols as well as options for low cost methods for cloning verification and gene editing verification in planta. We hope that our toolkit helps bridge the gap between the recent biotechnological advancements in plant breeding that high income countries can access and the lack of those technologies in low and middle income countries.