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CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

Ronda C, Maury J, Jakočiunas T, Jacobsen SA, Germann SM, Harrison SJ, Borodina I, Keasling JD, Jensen MK, Nielsen AT - Microb. Cell Fact. (2015)

Bottom Line: Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae.Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

View Article: PubMed Central - PubMed

Affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. carro@biosustain.dtu.dk.

ABSTRACT

Background: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.

Results: Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes.

Conclusions: The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

No MeSH data available.


Related in: MedlinePlus

Overview of the biosynthetic pathway for β-carotene production. The carotenoid biosynthetic pathway can be reconstructed in S. cerevisiae by overexpression of the native GGPP synthase encoded by BTS1, and co-overexpression of the non-native bifunctional phytoene synthase/lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI of X. dendrorhous. HMG1 encodes the major HMG-CoA reductase activity in S. cerevisiae. ERG9 encodes a farnesyl-diphosphate farnesyl transferase (squalene synthase) that acts in the sterol biosynthesis pathway. IPP isopentenyl diphosphate, DMAP dimethylallyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl diphosphate.
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Fig2: Overview of the biosynthetic pathway for β-carotene production. The carotenoid biosynthetic pathway can be reconstructed in S. cerevisiae by overexpression of the native GGPP synthase encoded by BTS1, and co-overexpression of the non-native bifunctional phytoene synthase/lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI of X. dendrorhous. HMG1 encodes the major HMG-CoA reductase activity in S. cerevisiae. ERG9 encodes a farnesyl-diphosphate farnesyl transferase (squalene synthase) that acts in the sterol biosynthesis pathway. IPP isopentenyl diphosphate, DMAP dimethylallyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl diphosphate.

Mentions: As a proof of concept for the applicability of CrEdit for metabolic engineering, we used the well-established carotenoid biosynthetic pathway as a model. Carotenoids are part of the diverse group of natural compounds called isoprenoids, and are synthesized from precursors derived from the native mevalonic acid (MVA) pathway (Figure 2). The tHMG1 gene encodes a truncated HMG-CoA reductase, which has been shown to increase carbon flux through the pathway, leading to increased isoprenoid and carotenoid production [33, 34]. Therefore, we initially focused on introducing one copy of the tHMG1 overexpression cassette into the S. cerevisiae genome.Figure 2


CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

Ronda C, Maury J, Jakočiunas T, Jacobsen SA, Germann SM, Harrison SJ, Borodina I, Keasling JD, Jensen MK, Nielsen AT - Microb. Cell Fact. (2015)

Overview of the biosynthetic pathway for β-carotene production. The carotenoid biosynthetic pathway can be reconstructed in S. cerevisiae by overexpression of the native GGPP synthase encoded by BTS1, and co-overexpression of the non-native bifunctional phytoene synthase/lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI of X. dendrorhous. HMG1 encodes the major HMG-CoA reductase activity in S. cerevisiae. ERG9 encodes a farnesyl-diphosphate farnesyl transferase (squalene synthase) that acts in the sterol biosynthesis pathway. IPP isopentenyl diphosphate, DMAP dimethylallyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl diphosphate.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492099&req=5

Fig2: Overview of the biosynthetic pathway for β-carotene production. The carotenoid biosynthetic pathway can be reconstructed in S. cerevisiae by overexpression of the native GGPP synthase encoded by BTS1, and co-overexpression of the non-native bifunctional phytoene synthase/lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI of X. dendrorhous. HMG1 encodes the major HMG-CoA reductase activity in S. cerevisiae. ERG9 encodes a farnesyl-diphosphate farnesyl transferase (squalene synthase) that acts in the sterol biosynthesis pathway. IPP isopentenyl diphosphate, DMAP dimethylallyl diphosphate, GPP geranyl diphosphate, FPP farnesyl diphosphate, GGPP geranylgeranyl diphosphate.
Mentions: As a proof of concept for the applicability of CrEdit for metabolic engineering, we used the well-established carotenoid biosynthetic pathway as a model. Carotenoids are part of the diverse group of natural compounds called isoprenoids, and are synthesized from precursors derived from the native mevalonic acid (MVA) pathway (Figure 2). The tHMG1 gene encodes a truncated HMG-CoA reductase, which has been shown to increase carbon flux through the pathway, leading to increased isoprenoid and carotenoid production [33, 34]. Therefore, we initially focused on introducing one copy of the tHMG1 overexpression cassette into the S. cerevisiae genome.Figure 2

Bottom Line: Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae.Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

View Article: PubMed Central - PubMed

Affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. carro@biosustain.dtu.dk.

ABSTRACT

Background: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.

Results: Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes.

Conclusions: The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

No MeSH data available.


Related in: MedlinePlus