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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

Schematic overview of the CrEdit system. a Replicative vectors expressing gRNAs and cas9. b Targeted DSBs mediated by Cas9 endonuclease activity facilitate the integration of linearized donor plasmids by homologous recombination. Donor plasmids harbor the desired integration sequences flanked by homology arms. Selected intergenic safe harbor sites can be used for simultaneous integration of multiple genomic expression cassettes for pathway engineering.
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Fig1: Schematic overview of the CrEdit system. a Replicative vectors expressing gRNAs and cas9. b Targeted DSBs mediated by Cas9 endonuclease activity facilitate the integration of linearized donor plasmids by homologous recombination. Donor plasmids harbor the desired integration sequences flanked by homology arms. Selected intergenic safe harbor sites can be used for simultaneous integration of multiple genomic expression cassettes for pathway engineering.

Mentions: In order to increase the efficiency of targeted integration into the S. cerevisiae genome, we decided to combine the well-characterized genomic integration sites used in the EasyClone system with the RNA-guided endonuclease activity of Cas9. Initially, we tested two different designs for the system. In the first design, Cas9 was expressed from a constitutive promoter, PTEF1, on an ARS/CEN based vector, while the gRNA that targets Cas9 to the chosen EasyClone integration site was expressed from an episomal 2μ-based vector (Figure 1) [24]. In the second design, Cas9 was under the control of the inducible PCUP1 promoter and integrated in the genome, and the gRNA supplied on a linearized integrative vector. The first design was chosen for its versatile and recyclable aspects, while the second design was chosen for the possibility of controlling the expression of Cas9 and gRNAs at lower levels. Both types of gRNA carrier plasmids have been designed to enable a fast exchange of the gRNA expression cassettes via USER cloning. Thereby, it is possible to conveniently target a new locus by quick and easy single-step cloning of the gRNA plasmids [27]. Also, the USER-overhang system enables multiplexing of up to five gRNAs on one single plasmid [27].Figure 1


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)

Schematic overview of the CrEdit system. a Replicative vectors expressing gRNAs and cas9. b Targeted DSBs mediated by Cas9 endonuclease activity facilitate the integration of linearized donor plasmids by homologous recombination. Donor plasmids harbor the desired integration sequences flanked by homology arms. Selected intergenic safe harbor sites can be used for simultaneous integration of multiple genomic expression cassettes for pathway engineering.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic overview of the CrEdit system. a Replicative vectors expressing gRNAs and cas9. b Targeted DSBs mediated by Cas9 endonuclease activity facilitate the integration of linearized donor plasmids by homologous recombination. Donor plasmids harbor the desired integration sequences flanked by homology arms. Selected intergenic safe harbor sites can be used for simultaneous integration of multiple genomic expression cassettes for pathway engineering.
Mentions: In order to increase the efficiency of targeted integration into the S. cerevisiae genome, we decided to combine the well-characterized genomic integration sites used in the EasyClone system with the RNA-guided endonuclease activity of Cas9. Initially, we tested two different designs for the system. In the first design, Cas9 was expressed from a constitutive promoter, PTEF1, on an ARS/CEN based vector, while the gRNA that targets Cas9 to the chosen EasyClone integration site was expressed from an episomal 2μ-based vector (Figure 1) [24]. In the second design, Cas9 was under the control of the inducible PCUP1 promoter and integrated in the genome, and the gRNA supplied on a linearized integrative vector. The first design was chosen for its versatile and recyclable aspects, while the second design was chosen for the possibility of controlling the expression of Cas9 and gRNAs at lower levels. Both types of gRNA carrier plasmids have been designed to enable a fast exchange of the gRNA expression cassettes via USER cloning. Thereby, it is possible to conveniently target a new locus by quick and easy single-step cloning of the gRNA plasmids [27]. Also, the USER-overhang system enables multiplexing of up to five gRNAs on one single plasmid [27].Figure 1

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