CRISPR-based construction of a hexose-transport-deficient Saccharomyces cerevisiae strain and its toolkit. Saccharomyces cerevisiae strain:IMX1812

Hexose-transporters deficient yeast strains are very valuable testbeds for the study of hexose transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae EBY.VW4000, deletion of 21 transporters completely abolished resulted in the complete abolishment of hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic alterations. In the present study CRISPR Cas9 was used to delete the 21 hexose transporters in a S. cerevisiae strain from the CEN.PK family in three deletion rounds, using 11 single guide RNAs. The resulting strain, named CRISPR-Hxt0 was unable to consume glucose, even upon prolonged cultivation, but grew as well as its parent with a full set of hexose transporters on maltose. Karyotyping and whole genome sequencing using both Illumina and Oxford Nanopore technologies revealed the absence of chromosomal and genetic alterations, besides HXT genes deletion, in CRISPR-Hxt0.This study not only provides a new, ‘genetically unaltered’ hexose-transporter deficient strain, but also supplies a CRISPR toolkit and facile method to remove all hexose transporters in most S. cerevisiae laboratory strains in three transformation rounds.