Sugarcane genome editing using CRISPR/Cas9 for targeted mutagenesis of ethylene biosynthesis

• Grant number: 17/09817-0
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: November 01, 2017
• End date: October 31, 2018
• Field of knowledge: Biological Sciences - Genetics - Plant Genetics
• Principal Investigator: Marcelo Menossi Teixeira
• Grantee: Lucia Mattiello
• Supervisor: Fredy Altpeter
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: University of Florida, Gainesville (UF), United States
• Associated to the scholarship: 15/23789-4 - EVALUATION OF THE ROLE OF GENES ENCODING ACC SINTHASE IN THE DEVELOPMENT OF SUGARCANE PLANTS, BP.PD

Abstract

Nucleases, like CRISPR/Cas9, can be designed to create double strand breaks (DSB) in specific DNA sequences. These DBS will stimulate the plant cell's DNA repair system that may result in frameshift mutations due to deletions and insertions typical of this error prone process, producing loss of function mutants. One major advantage of CRISPR/Cas9 is its multiplexing capacity, which allows efficient co-mutation of multiple targets by co-delivery of the Cas9 enzyme and multiple guide RNAs. Although ethylene is a key component for maturation on several plant species, the role of this hormone in sugarcane remains elusive. CRISPR/Cas9 is the most versatile genome editing tool and will be used in this project to suppress three genes encoding the enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS). To this end, we propose: (a) target selection and gRNA design and synthesis; (b) transfer of CRISPR constructs into transgenic sugarcane for gene editing; (d) molecular characterization of genome edited plants; (e) evaluation of the impacts in ACS genes expression material, and (f): evaluation of ethylene production in the ACS-edited sugarcane plants. After the one-year period at the University of Florida, the plants will be shipped to Brazil and physiological evaluations will be performed. (AU)