Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

Antony, Jacob S.; Hinz, John M.; Wyrick, John J.

doi:10.3389/fbioe.2022.924914

Cited by 11 publications

(9 citation statements)

References 212 publications

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“…In the cases of RRT5 and PDR12 , we could not find yeast strains in the population carrying alleles with just the SNPs needed, so we had to introduce them with CRISPR-Cas9 technology. This was technically difficult because the target region must include the SNP to be changed, limiting the region for gRNA design, which is key to correctly applying this technique [ 40 ]. This leads to the fact that, in the case of PDR12 , only one of the two desired point mutations could be made (4393G>A), which corresponds to a conservative missense mutation from valine to isoleucine (V1465I) in the amino acid sequence of Pdr12 (Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains

et al. 2023

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For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the “1002 Yeast Genomes Project” population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

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Section: Resultsmentioning

confidence: 99%

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains

et al. 2023

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“…The advent of new technologies, such as CRISPR-Cas, provides brand new ideas and greater operable space. The selective uniting of multiple gene editing technologies and the combination with efficient expression systems have significantly improved the precision and accuracy of biotechnology and applications, and the scope of function is evolving from single to multi-gene and from low to high throughput ( Yilmaz, 2021 ; Antony et al, 2022 ). In the future, other types of genetic modifications may be achieved, such as gene motivation and gene interference with a controlled degree of expression, exploring more orthologues of functional enzymes within organisms, as well as cross-species gene mapping.…”

Section: Discussionmentioning

confidence: 99%

Establishment, optimization, and application of genetic technology in Aspergillus spp.

et al. 2023

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Aspergillus is widely distributed in nature and occupies a crucial ecological niche, which has complex and diverse metabolic pathways and can produce a variety of metabolites. With the deepening of genomics exploration, more Aspergillus genomic informations have been elucidated, which not only help us understand the basic mechanism of various life activities, but also further realize the ideal functional transformation. Available genetic engineering tools include homologous recombinant systems, specific nuclease based systems, and RNA techniques, combined with transformation methods, and screening based on selective labeling. Precise editing of target genes can not only prevent and control the production of mycotoxin pollutants, but also realize the construction of economical and efficient fungal cell factories. This paper reviewed the establishment and optimization process of genome technologies, hoping to provide the theoretical basis of experiments, and summarized the recent progress and application in genetic technology, analyzes the challenges and the possibility of future development with regard to Aspergillus.

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“…Scale increases in the production line of null segregants also increase the rate of production of both unintended outcomes from intended changes and unintended outcomes from unintended changes ( Kawall et al, 2020 ; Antony, Hinz, and Wyrick, 2022 ; Chu and Agapito-Tenfen, 2022 ). The Asilomar scientists were aware of the risk amplification with scale.…”

Section: Technological Risk Perspectivesmentioning

confidence: 99%

Are null segregants new combinations of heritable material and should they be regulated?

et al. 2023

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Through genome editing and other techniques of gene technology, it is possible to create a class of organism called null segregants. These genetically modified organisms (GMOs) are products of gene technology but are argued to have no lingering vestige of the technology after the segregation of chromosomes or deletion of insertions. From that viewpoint regulations are redundant because any unique potential for the use of gene technology to cause harm has also been removed. We tackle this question of international interest by reviewing the early history of the purpose of gene technology regulation. The active ingredients of techniques used for guided mutagenesis, e.g., site-directed nucleases, such as CRISPR/Cas, are promoted for having a lower potential per reaction to create a hazard. However, others see this as a desirable industrial property of the reagents that will lead to genome editing being used more and nullifying the promised hazard mitigation. The contest between views revolves around whether regulations could alter the risks in the responsible use of gene technology. We conclude that gene technology, even when used to make null segregants, has characteristics that make regulation a reasonable option for mitigating potential harm. Those characteristics are that it allows people to create more harm faster, even if it creates benefits as well; the potential for harm increases with increased use of the technique, but safety does not; and regulations can control harm scaling.

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Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

Cited by 11 publications

References 212 publications

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains

Establishment, optimization, and application of genetic technology in Aspergillus spp.

Are null segregants new combinations of heritable material and should they be regulated?

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