Establishment of transgene-free and bipartite surrogate gene editing strategies via CRISPR base editing and Prime editing systems in sugarcane

• Grant number: 24/02180-0
• Support Opportunities: Regular Research Grants
• Start date: August 01, 2024
• End date: July 31, 2026
• Field of knowledge: Agronomical Sciences - Agronomy - Crop Science
• Agreement: CNPq
• Principal Investigator: Marcelo Menossi Teixeira
• Grantee: Marcelo Menossi Teixeira
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 22/04006-2 - Center for Plant Molecular Breeding (CeM²P), AP.PCPE
• Associated scholarship(s): 24/13410-7 - Establishment of transgene-free and bipartite surrogate gene editing strategies via base editing and Prime editing CRISPR systems in sugarcane., BP.JD

Abstract

The challenges of the growing demand for food and to reduce greenhouse gas emissions require urgency in the development and commercial approval of improved cultivars. Sugarcane is one of the predominant crops in Brazil and one of the most promising sources of renewable energy today. Therefore, the establishment of a transgene-free CRISPR/Cas9-mediated gene editing protocol in sugarcane is an important topic in the economic and environmental spheres. However, transgene-free gene editing protocols present low efficiency due to the absence of a selective marker that helps the identification of edited plants during tissue culture. Recently, cytidine base editing (CBE), adenosine base editing (ABE) and Prime editing (PE) approaches, which are derived from the CRISPR/Cas9 system, have been used to generate selective markers in plants through installation of mutations that provide tolerance to herbicides. However, the application of the CRISPR/Cas9 system is still incipient in sugarcane and reports of the application of the CBE and PE systems are, to date, non-existent. Therefore, this proposal aims to test the CBE and PE systems in sugarcane with a view to producing edited and transgene-free plants. Specifically, the A96V and W574L mutations, which confer tolerance to the Imidazolinones class of herbicides, will be installed in the sugarcane ScALS gene via the CBE and PE systems, respectively. In parallel, this project also proposes the establishment of a surrogate strategy based on the ABE system for facilitated commercial approval of edited plants containing only the NPTII selective marker gene in their genome, already widely used in commercially released materials. If successful, the activities of this proposal will represent a major advance in the application of the CRISPR/Cas9 system, in the development of improved cultivars, new devices for weed management and new tools for studying gene function in sugarcane. (AU)