Abstract
An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial “starter” strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8–12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts.
Highlights
While for laboratory strains of Saccharomyces cerevisiae several molecular methods have allowed extensive interpretation of gene functionality, industrial and wild yeast strains are still poorly studied; the genetic manipulation of latter yeasts can be time consuming because of they are usually recalcitrant to some molecular techniques and they are characterized by complex genomes
The results presented here show the successful editing of the CAN1 gene of two S. cerevisiae wine yeast strains using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system that consists of three elements: two expression vectors carrying the Cas9 gene and the gRNA, respectively, and a donor dsDNA fragment
We outline a strategy to modify wine yeasts with the CRISPR/Cas9 system, an efficient, cheap and easy-to-use tool for genome editing that allows the simultaneous modification of all the alleles of a target gene
Summary
While for laboratory strains of Saccharomyces cerevisiae several molecular methods have allowed extensive interpretation of gene functionality, industrial and wild yeast strains are still poorly studied; the genetic manipulation of latter yeasts can be time consuming because of they are usually recalcitrant to some molecular techniques and they are characterized by complex genomes (i.e., diploid and polyploid species). For this reason, the development of a rapid and efficient gene-targeting system based on the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein (CRISPR-Cas9) system is gaining attention in several industrial fields. The crRNAs hybridize with transactivating CRISPR RNAs (tracrRNAs) and the resulting crRNA/tracrRNA complex acts as a guide for the endonuclease Cas, which cleaves invading nucleic acid sequences (Brouns, 2012; DiCarlo et al, 2013)
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