Glutathione (GSH), a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9-associated-system (CRISPR-Cas), in which gRNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1b/FGP. As a result, the prototrophic strain W303-1b/FGPPT showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1b/FGPPT that might have great interests in industrial fermentation.