A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains

Abstract

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.

Figure 1.

(A) Overview of the chromosomal localization of the deleted hexose transporters. Genes indicated with the same color were removed in the same deletion round. Red, first round; blue, second round; green, third round. (B) Deletion strategy. The scissors indicate the gene targeted by SpCas9 editing. The circled numbers indicate the sgRNA used to guide SpCas9 for editing.

Figure 2.

Physiological characterization of the CRISPR-Hxt⁰ strain IMX1812 and its ancestors. (A) Growth in aerobic shake-flasks with chemically defined medium using maltose as sole carbon source. CEN.PK2-1C: control strain without SpCas9, IMX672: control strain constitutively expressing SpCas9 from its genomic DNA, IMX1812: CRISPR-Hxt⁰ strain and EBY.VW4000: Hxt⁰ strain from Wieczorke et al. (1999). All strains are quadruple auxotrophs and were supplied with uracil, leucine, histidine and tryptophan. Data represent the average and mean deviation of biological duplicates for each strain and growth condition. The asterisk indicates a P value below 0.05 (Student t-test) as compared to the control strain CEN.PK2-1C. (B) Growth on chemically defined medium with various carbon sources. IMX1521 and IMX1541 were obtained after the first and second transformation round, respectively. The pictures were taken after 2 days of incubation at 30°C.

Figure 3.

Genomic characterization of the CRISPR-Hxt⁰ strain IMX1812, its ancestors and EBY.VW4000. (A) Karyotyping using pulse-field electrophoresis. Red arrows indicate size decrease of chromosomes as consequence of CRISPR-based hexose transporter genes deletion (Supplemental Material 4). Blue boxes indicate polymorphisms in EBY.VW4000 as compared to the control strains CEN.PK113-7D. (B) Circos plot comparing the chromosomes of EBY.VW4000 (left side of the circle, indicated in red) and of the control strain CEN.PK113-7D (right side of the circle, indicated in blue). (C) Circos plot comparing the chromosomes of IMX1812 (left side of the circle, indicated in red) and of the control strain CEN.PK113-7D (right side of the circle, indicated in blue). (D) Chromosome copy number variation of IMX1812 based on Nanopore sequencing data. Blue dots represent the parental strain CEN.PK2-1C and red dots the Hxt⁰ strain IMX1812. (E) Cellular DNA content measurement by flow cytometry. CEN.PK113-7D and CEN.PK122 are the haploid and diploid controls, respectively.