A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains.
Biological Transport
CRISPR-Associated Protein 9
/ metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Deletion
Gene Editing
/ methods
Genotype
Hexoses
/ metabolism
Karyotyping
Monosaccharide Transport Proteins
/ genetics
Mycological Typing Techniques
Saccharomyces cerevisiae
/ genetics
Sequence Analysis, DNA
Journal
FEMS yeast research
ISSN: 1567-1364
Titre abrégé: FEMS Yeast Res
Pays: England
ID NLM: 101085384
Informations de publication
Date de publication:
01 01 2019
01 01 2019
Historique:
received:
15
07
2018
accepted:
01
10
2018
pubmed:
5
10
2018
medline:
13
7
2019
entrez:
5
10
2018
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 30285096
pii: 5114578
doi: 10.1093/femsyr/foy107
pmc: PMC6217715
doi:
Substances chimiques
Hexoses
0
Monosaccharide Transport Proteins
0
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
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