A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9

Mans, Robert; Wijsman, Melanie; Daran‐Lapujade, Pascale; Daran, Jean‐Marc

doi:10.1093/femsyr/foy063

Cited by 26 publications

(17 citation statements)

References 14 publications

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“…The double sgRNA method for CRISPR/ Sp Cas9-mediated deletions based on pROS plasmid series (Mans et al. 2015 , 2018 ) was used to construct several sgRNA plasmids. The guide RNA sequences were selected to perform the 21 hexose transporter deletions in a minimal number of transformation rounds.…”

Section: Methodsmentioning

confidence: 99%

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

Wijsman

Swiat

Marques

et al. 2018

FEMS Yeast Research

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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.

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Section: Methodsmentioning

confidence: 99%

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

Wijsman

Swiat

Marques

et al. 2018

FEMS Yeast Research

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“…Plasmids carrying two copies of the same gRNA were cloned by in vitro Gibson assembly as previously described (70). In brief, an oligo carrying the 20 bp target sequence and homology to the backbone plasmid was used to amplify the fragment carrying the 2µm origin of replication sequence by using pROS13 as template.…”

Section: Methodsmentioning

confidence: 99%

Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies inSaccharomyces cerevisiae

Perli

Moonen

Broek

et al. 2020

Preprint

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AbstractQuantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B-vitamins. Previous work demonstrated that, in S. cerevisiae CEN.PK113-7D, requirements for biotin could be eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA) or thiamine were omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B-vitamins. In all evolution lines, specific growth rates reached at least 90 % of the growth rate observed in SM supplemented with a complete B-vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid or pABA. Reaching similar results in SM lacking thiamine, pyridoxine or pantothenate required over 300 generations of selective growth. The genomes of evolved single-colony isolates were re-sequenced and, for each B-vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth were selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B-vitamin, introduction of a small number of mutations sufficed to achieve substantially a increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.ImportanceMany strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid and inositol. However, omission of these B-vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B-vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotype by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step towards development of S. cerevisiae strains that exhibit fast growth on cheap, fully mineral media that only require complementation with a carbon source, thereby reducing costs, complexity and contamination risks in industrial yeast fermentation processes.

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“…Plasmids carrying two copies of the same gRNA were cloned by in vitro Gibson assembly as previously described (72). In brief, an oligo carrying the 20 bp target sequence and homology on October 1, 2020 by guest http://aem.asm.org/…”

Section: Plasmids Cloningmentioning

confidence: 99%

Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae

Perli

Moonen

Broek

et al. 2020

Appl Environ Microbiol

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Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM. IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.

show abstract

A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9

Cited by 26 publications

References 14 publications

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficientSaccharomyces cerevisiaestrains

Adaptive laboratory evolution and reverse engineering of single-vitamin prototrophies inSaccharomyces cerevisiae

Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae

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