Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

Shan, Lu; Dai, Zhendong; Wang, Qinhong

doi:10.3389/fbioe.2021.765396

Cited by 16 publications

(16 citation statements)

References 68 publications

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“…Other pol III promoters like RPR1 can also be used to express sgRNAs in yeast 38,39 . All these promoters are pol III promoters that worked well with high efficiency.…”

Section: Discussionmentioning

confidence: 99%

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Episomal editing of synthetic constructs in yeast using CRISPR

Zhao

Coelho

Lauer

et al. 2022

Preprint

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Use of synthetic genomics to design and build ″big″ DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. S. cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, efficiently and precisely introducing designer variations to episomal assemblies remains challenging. Here, we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, for rapid engineering of mammalian DNA constructs larger than 100 kb. We demonstrate that editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes with CRISPR. After optimizing CREEPY for episomal editing, we achieve efficient simplex and multiplex editing as demonstrated by engineering a mouse Sox2-harboring episome.

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“…Other pol III promoters like RPR1 can also be used to express sgRNAs in yeast 38,39 . All these promoters are pol III promoters that worked well with high efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…For sgRNA expression, the SNR52 promoter and endogenous tRNA were used in this study. Other pol III promoters like RPR1 can also be used to express sgRNAs in yeast 38, 39 . All these promoters are pol III promoters that worked well with high efficiency.…”

Section: Discussionmentioning

confidence: 99%

Episomal editing of synthetic constructs in yeast using CRISPR

Zhao

Coelho

Lauer

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…With such synthetic biology strategies, engineering of S. cerevisiae , Y. lipolytica or other yeasts to produce Lepidopteran sex pheromones has achieved considerable success. With further development and application of advanced yeast tools, such as high throughput screening strategy ( Tan et al, 2020 ), metabolic mass transfer strategy ( Xue et al, 2021 ), and efficient gene editing tools for non-conventional oleaginous yeasts ( Shan et al, 2021 ), large-scale commercial production of sex pheromones in engineered yeasts with a high titer, rate and yield would be possible, and will also facilitate moth-mating disruption using biosynthetic sex pheromones in a cost-efficient manner. In the future, the application of yeast-based sex pheromones will lead to eco-friendly agriculture with a green pest control strategy.…”

Section: Conclusion and Future Perspectivementioning

confidence: 99%

Developments in Fatty Acid-Derived Insect Pheromone Production Using Engineered Yeasts

Miao

et al. 2021

Front. Microbiol.

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The use of traditional chemical insecticides for pest control often leads to environmental pollution and a decrease in biodiversity. Recently, insect sex pheromones were applied for sustainable biocontrol of pests in fields, due to their limited adverse impacts on biodiversity and food safety compared to that of other conventional insecticides. However, the structures of insect pheromones are complex, and their chemical synthesis is not commercially feasible. As yeasts have been widely used for fatty acid-derived pheromone production in the past few years, using engineered yeasts may be promising and sustainable for the low-cost production of fatty acid-derived pheromones. The primary fatty acids produced by Saccharomyces cerevisiae and other yeasts are C16 and C18, and it is also possible to rewire/reprogram the metabolic flux for other fatty acids or fatty acid derivatives. This review summarizes the fatty acid biosynthetic pathway in S. cerevisiae and recent progress in yeast engineering in terms of metabolic engineering and synthetic biology strategies to produce insect pheromones. In the future, insect pheromones produced by yeasts might provide an eco-friendly pest control method in agricultural fields.

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“…al. 2019;Román et al 2019b;Schuster and Kahmann 2019;Morio et al 2020;Ouedraogo and Tsang 2020;Shan et al 2021), including numerous non-albicans Candida species(Santana and O'Meara;Enkler et al 2016;Lombardi et al 2017Lombardi et al , 2019Zoppo et al 2019;Maroc and Fairhead 2019;Uthayakumar et al 2021;Ennis et al 2021), and other prominent pathogens such as…”

mentioning

confidence: 99%

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

Gervais

Bella

Wensing

et al. 2022

Preprint

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For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness genetic tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans. Our CRISPRa system exploits a nuclease-dead dCas9 fused to the tripartite activator complex VP64-p65-Rta (VPR), and a Golden Gate site for efficient guide RNA cloning to overexpress genes of interest. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust overexpression. We further demonstrate the application of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these traits. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

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Advances and Opportunities of CRISPR/Cas Technology in Bioengineering Non-conventional Yeasts

Cited by 16 publications

References 68 publications

Episomal editing of synthetic constructs in yeast using CRISPR

Episomal editing of synthetic constructs in yeast using CRISPR

Developments in Fatty Acid-Derived Insect Pheromone Production Using Engineered Yeasts

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

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