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

• Grant number: 24/13410-7
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• Start date: August 01, 2024
• End date: July 31, 2025
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Agreement: CNPq
• Principal Investigator: Marcelo Menossi Teixeira
• Grantee: Vitor Favaretto Pinoti
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 24/02180-0 - Establishment of transgene-free and bipartite surrogate gene editing strategies via CRISPR base editing and Prime editing systems in sugarcane, AP.R

Abstract

The rapid development and commercial approval of improved cultivars aimed at reducing greenhouse gas emissions and increasing productivity are imperative to meet growing food demand. Sugarcane is one of the most predominant crops in Brazil and is considered one of the most promising renewable energy sources today. Therefore, the establishment of a gene editing protocol mediated by CRISPR/Cas9 and free of transgenes in sugarcane is an important topic in the economic and environmental subjects. 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 to produce transgene-free edited plants. Specifically, the A96V and W574L mutations, which confer tolerance to the Imidazolinones classes 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. 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.