CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

Löbs, Ann-Kathrin; Engel, Ronja; Schwartz, Cory; Flores, Andrew; Wheeldon, Ian

doi:10.1186/s13068-017-0854-5

Cited by 99 publications

(109 citation statements)

References 60 publications

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“…The widespread adoption of type II CRISPR–Cas9 systems for genome editing has made less genetically tractable organisms more accessible (Jacobs et al, ; Pohl et al, ; Schwartz et al, ; Löbs, Engel, Schwartz, Flores, & Wheeldon, ). CRISPRi system have extended these capabilities to targeted transcriptional regulation (Qi, Larson, et al, ; Singh et al, ).…”

Section: Discussionmentioning

confidence: 99%

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

Schwartz

Frogue

Ramesh

et al. 2017

Biotech & Bioengineering

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107

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In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.

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Section: Discussionmentioning

confidence: 99%

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

Schwartz

Frogue

Ramesh

et al. 2017

Biotech & Bioengineering

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107

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“…Combined with CRISPR-Cas9 genome editing ( Fig. 1) (6)(7)(8)(9), these results establish a full set of tools for use of K. marxianus as a synthetic biology host and future exploration of its biology on a genome-wide scale.…”

Section: Discussionmentioning

confidence: 77%

“…Therefore, we inactivated genes by CRISPR NHEJ to decrease ethanol fermentation (ADH genes) (26), and ethyl acetate production (ATF genes) (7) as well as ester biosynthesis (EHT1) (7,27) and glycerol biosynthesis (GPP1). We also inactivated genes involved in β-oxidation (PEX10 and MFE1) (23) ( Fig.…”

Section: Strain Engineering For Higher Lipid Productionmentioning

confidence: 99%

“…These tools would enable rapid strain development, by generating genetic diversity and facilitating stacking of industrially important traits. The CRISPR-Cas9 gene editing system has been used in many yeasts including S. cerevisiae, S. pombe, Y. lipolytica, K. lactis and recently K. marxianus (6)(7)(8)(9). Genome editing in K. marxianus should allow manipulation of known genetic targets.…”

Section: Introductionmentioning

confidence: 99%

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Kluyveromyces marxianus as a robust synthetic biology platform host

Cernak

Estrela

Poddar

et al. 2018

Preprint

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Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traitsthermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

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“…However, these techniques have not yet been widely translated to non-model and other industrially and medically relevant organisms. The widespread adoption of CRISPR-Cas9 promises to enable genome-wide engineering and screening for a vast array of hosts 3,5,12,[42][43][44][45] . Here, we designed and applied such a system for functional genomic screening and nonnative phenotype engineering in the yeast Y. lipolytica.…”

Section: Discussionmentioning

confidence: 99%

Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

Schwartz

Cheng

Evans

et al. 2018

Preprint

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Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. To address this limitation, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled highthroughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which functional determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified mutations that led to high lipid accumulation and eliminated pseudohyphal morphology.

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CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

Cited by 99 publications

References 60 publications

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

Kluyveromyces marxianus as a robust synthetic biology platform host

Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

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