Antisense oligonucleotide and CRISPR/CAS strategy for caffeine reduction in Coffea arabica

Abstract

Coffee is the most important agricultural commodity in the world, being distributed and traded globally in a multi-million-dollar industry. Of the compounds present in coffee beans, caffeine is one of the best known and noticed due its physiological effects on humans and its role in the physiology and life cycle on coffee plant. The caffeine content of coffee beans differs between coffee Coffea species: C. arabica beans contain ~1.2% caffeine, C. canephora ~2.4% and C. eugenioides ~0.3-0.8%. Some studies show that secondary metabolites such as caffeine and chlorogenic acid produced by several plants are important in its ability to defend itself against pathogens and competing plants. However, some breeding programs are focused on producing coffee plants without or reduced caffeine content. Naturally decaffeinated C. arabica plants were identified in the germplasm collection of the Instituto Agronomico de Campinas (IAC). It was seen that AC1 genotype accumulated the compound theobromine but not caffeine, which suggests an alteration in the caffeine synthase enzyme, responsible for theobromine methylation and subsequent caffeine formation. In this context, our work aims to silence the caffeine synthase gene in C. arabica, via HDR (Homology Directed Repair) mediated by CRISPR/cas, using embryogenic calli and somatic embryogenesis as a regenerated method; and also to test ASO (antisense oligonucleotides) technology to reduce caffeine contents evaluating the relationship of these silencing and decrease in plant caffeine levels and studying the possible effects of a decaffeinated plant in relation to biotic stress; such as that provided by the interaction with the fungus that causes coffee rust, Hemileia Vastatrix.