CRISPR/CAS9 system as a valuable genome editing tool applicate to wine yeasts

Póster presentado en la 3rd International Conference on Microbial Diversity (Microbial Diversity 2015. The challenge of Complexity), celebrada en Perugia del 27 al 29 de octubre de 2015.
Saccharomyces cerevisiae has an extensive repertoire of molecular methods for the genetic analysis of laboratory strains. Although this has widely contributed to the interpretation of gene functionality within haploid genomes, the genetic basis of desirable metabolic pathways in industrial diploid and polyploid yeast strain is still poorly understood. The genetic engineering of industrial yeasts is currently undergoing major changes due to the development of a marker-free, high-throughput, and multiplexed genome editing approach inspired to the bacterial immune systems: the “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) immune system”. CRISPR/Cas9 allows the knockout or the tuning of expression of specific targeted genes and pathways. The system requires an endonuclease (Cas9) that is guided to a specific DNA sequence by a guide RNA (gRNA). The assembling of a ribonucleoprotein complex (Cas9/gRNA) can produce cuts in specific sites of eukaryotic genomes and generates different combinations of gene deletions through a single transformation event. CRISPR/Cas9 is gaining significant attention because of its biotechnological applications and its potential to boost innovation. The wine industry could particularly gain an advantage from this engineering system; indeed, molecular studies should help understanding the contribution of Saccharomyces and non-Saccharomyces species to several wine features such as those linked to wine quality and safety (aroma and off-flavours compounds, ethanol and glycerol production, sulphur dioxide resistance, toxic compound formation, etc.). In this study we outline a strategy to modify wine yeasts by CRISPR/Cas9 approach. To prove the robustness of this system and to offers an engineering pipeline for further gene editing in specific metabolic pathways relevant for the wine production, two commercial strains of S. cerevisiae (EC1118 and AWRI 796) have been genetically engineered in the arginine degradation pathway to generate strains with a reduced urea and ethyl-carbamate production.