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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

Integration efficiency of tHMG1 at locus X-2 using different lengths of homology arms. a Overview of the donor DNA fragment bearing tHMG1 with differently sized homology arms. b Integration efficiency of the CrEdit system with genomic inducible Cas9 and integrative gRNA. S. cerevisiae strain ST1011 harboring PCUP1-cas9 was induced with Cu2+ 2 h prior to transformation start, and then co-transformed with (left, –gRNA) linearized empty vector pCfB257 and linearized donor DNA encoding tHMG1 (for details of donor DNA see Additional file 1), or (right, +gRNA) the linearized integrative gRNA vector pCfB2831 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. c Integration efficiency of the CrEdit system with plasmid-based Cas9 and gRNA. S. cerevisiae strain TC-3 harboring PTEF1-cas9 on the centromeric plasmid pCfB1767 was co-transformed with (left, −gRNA) empty vector pCfB2999 and linearized donor DNA encoding tHMG1, or (right, +gRNA) the episomal gRNA vector pCfB3020 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. Only +gRNA colonies were analyzed since no URA+ clones were obtained in the absence of gRNA. The experiment was repeated twice and error bars represent 95% confidence intervals. NA not analyzed.
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Fig3: Integration efficiency of tHMG1 at locus X-2 using different lengths of homology arms. a Overview of the donor DNA fragment bearing tHMG1 with differently sized homology arms. b Integration efficiency of the CrEdit system with genomic inducible Cas9 and integrative gRNA. S. cerevisiae strain ST1011 harboring PCUP1-cas9 was induced with Cu2+ 2 h prior to transformation start, and then co-transformed with (left, –gRNA) linearized empty vector pCfB257 and linearized donor DNA encoding tHMG1 (for details of donor DNA see Additional file 1), or (right, +gRNA) the linearized integrative gRNA vector pCfB2831 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. c Integration efficiency of the CrEdit system with plasmid-based Cas9 and gRNA. S. cerevisiae strain TC-3 harboring PTEF1-cas9 on the centromeric plasmid pCfB1767 was co-transformed with (left, −gRNA) empty vector pCfB2999 and linearized donor DNA encoding tHMG1, or (right, +gRNA) the episomal gRNA vector pCfB3020 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. Only +gRNA colonies were analyzed since no URA+ clones were obtained in the absence of gRNA. The experiment was repeated twice and error bars represent 95% confidence intervals. NA not analyzed.

Mentions: In order to test the efficiency of the two different CrEdit designs, we decided to test single integration of donor DNA with differently sized homology arms. As donor we used an EasyClone integrative plasmid containing tHMG1 with homology arms specific for intergenic site X-2 (Figure 3a) [15]. The integration efficiencies of all experiments are shown in Additional file 1: Table S1. We first tested the integration efficiency of using integrative gRNA in combination with a S. cerevisiae strain harboring genomic Cas9 under the control of the PCUP1 promoter. Cas9 expression was induced by addition of Cu2+ 2 h before transformation. We then co-transformed this Cas9-expressing strain with the specific donor DNA carrying tHMG1 with homology arms of 500, 110 or 60 bp length for site X-2, and the integrative gRNA targeting site X-2. An empty vector backbone without gRNA was used as a control. The resulting transformants were plated onto medium selecting for Cas9, the gRNA and the donor selection marker. We then analyzed the genotype of at least 16 colonies per condition to check for correct insertion at site X-2. When relying solely on intrinsic homologous recombination, the measured efficiency of correct integration at site X-2 was 70% with homology arms of approximately 500 bp (Figure 3b, left panel, −gRNA). As expected, the efficiency of correct integration was found to decrease significantly when using shorter arms with lengths of either 110 or 60 bp (Figure 3b, left panel, −gRNA). However, when the gRNA targeting X-2 was expressed, close to 100% successful integration was obtained at site X-2, regardless of the length of the homology arms (Figure 3b, left panel, +gRNA). Interestingly, when using the plasmid-based gRNA/Cas9 system and in the absence of gRNA, 100% correct integrants could only be obtained using 500 bp homology arms. Furthermore, and only in that condition, a low number of transformants was obtained on plates, which points towards a negative effect of cas9 expression on cells when expressed from the constitutive strong TEF1 promoter and in the absence of gRNA. Ryan et al. reported a decreased fitness of yeast strains expressing cas9 from the strong TDH3 promoter [26], while Mans et al. reported that the constitutive expression of cas9 from the genome and the TEF1 promoter does not affect the maximal specific growth rate on glucose based synthetic media [28]. In light of these results, a more detailed study of the impact of cas9 expression levels on yeast cell physiology and especially the HR machinery is of interest. Still, 100% correct integrants were obtained in the presence of gRNA for all sizes of homology arms (Figure 3c, left panel), suggesting that the plasmid-based gRNA/Cas9 system also is very efficient.Figure 3


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)

Integration efficiency of tHMG1 at locus X-2 using different lengths of homology arms. a Overview of the donor DNA fragment bearing tHMG1 with differently sized homology arms. b Integration efficiency of the CrEdit system with genomic inducible Cas9 and integrative gRNA. S. cerevisiae strain ST1011 harboring PCUP1-cas9 was induced with Cu2+ 2 h prior to transformation start, and then co-transformed with (left, –gRNA) linearized empty vector pCfB257 and linearized donor DNA encoding tHMG1 (for details of donor DNA see Additional file 1), or (right, +gRNA) the linearized integrative gRNA vector pCfB2831 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. c Integration efficiency of the CrEdit system with plasmid-based Cas9 and gRNA. S. cerevisiae strain TC-3 harboring PTEF1-cas9 on the centromeric plasmid pCfB1767 was co-transformed with (left, −gRNA) empty vector pCfB2999 and linearized donor DNA encoding tHMG1, or (right, +gRNA) the episomal gRNA vector pCfB3020 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. Only +gRNA colonies were analyzed since no URA+ clones were obtained in the absence of gRNA. The experiment was repeated twice and error bars represent 95% confidence intervals. NA not analyzed.
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Fig3: Integration efficiency of tHMG1 at locus X-2 using different lengths of homology arms. a Overview of the donor DNA fragment bearing tHMG1 with differently sized homology arms. b Integration efficiency of the CrEdit system with genomic inducible Cas9 and integrative gRNA. S. cerevisiae strain ST1011 harboring PCUP1-cas9 was induced with Cu2+ 2 h prior to transformation start, and then co-transformed with (left, –gRNA) linearized empty vector pCfB257 and linearized donor DNA encoding tHMG1 (for details of donor DNA see Additional file 1), or (right, +gRNA) the linearized integrative gRNA vector pCfB2831 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. c Integration efficiency of the CrEdit system with plasmid-based Cas9 and gRNA. S. cerevisiae strain TC-3 harboring PTEF1-cas9 on the centromeric plasmid pCfB1767 was co-transformed with (left, −gRNA) empty vector pCfB2999 and linearized donor DNA encoding tHMG1, or (right, +gRNA) the episomal gRNA vector pCfB3020 targeting X-2 and linearized donor DNA encoding tHMG1.Left panel Efficiency of targeted integration at site X-2 when selecting for donor DNA after transformation. Middle panel Efficiency of marker gene integration when not selecting for donor DNA after transformation. Right panel Frequency of correct integration at site X-2 determined by genotyping of URA+ colonies. Only +gRNA colonies were analyzed since no URA+ clones were obtained in the absence of gRNA. The experiment was repeated twice and error bars represent 95% confidence intervals. NA not analyzed.
Mentions: In order to test the efficiency of the two different CrEdit designs, we decided to test single integration of donor DNA with differently sized homology arms. As donor we used an EasyClone integrative plasmid containing tHMG1 with homology arms specific for intergenic site X-2 (Figure 3a) [15]. The integration efficiencies of all experiments are shown in Additional file 1: Table S1. We first tested the integration efficiency of using integrative gRNA in combination with a S. cerevisiae strain harboring genomic Cas9 under the control of the PCUP1 promoter. Cas9 expression was induced by addition of Cu2+ 2 h before transformation. We then co-transformed this Cas9-expressing strain with the specific donor DNA carrying tHMG1 with homology arms of 500, 110 or 60 bp length for site X-2, and the integrative gRNA targeting site X-2. An empty vector backbone without gRNA was used as a control. The resulting transformants were plated onto medium selecting for Cas9, the gRNA and the donor selection marker. We then analyzed the genotype of at least 16 colonies per condition to check for correct insertion at site X-2. When relying solely on intrinsic homologous recombination, the measured efficiency of correct integration at site X-2 was 70% with homology arms of approximately 500 bp (Figure 3b, left panel, −gRNA). As expected, the efficiency of correct integration was found to decrease significantly when using shorter arms with lengths of either 110 or 60 bp (Figure 3b, left panel, −gRNA). However, when the gRNA targeting X-2 was expressed, close to 100% successful integration was obtained at site X-2, regardless of the length of the homology arms (Figure 3b, left panel, +gRNA). Interestingly, when using the plasmid-based gRNA/Cas9 system and in the absence of gRNA, 100% correct integrants could only be obtained using 500 bp homology arms. Furthermore, and only in that condition, a low number of transformants was obtained on plates, which points towards a negative effect of cas9 expression on cells when expressed from the constitutive strong TEF1 promoter and in the absence of gRNA. Ryan et al. reported a decreased fitness of yeast strains expressing cas9 from the strong TDH3 promoter [26], while Mans et al. reported that the constitutive expression of cas9 from the genome and the TEF1 promoter does not affect the maximal specific growth rate on glucose based synthetic media [28]. In light of these results, a more detailed study of the impact of cas9 expression levels on yeast cell physiology and especially the HR machinery is of interest. Still, 100% correct integrants were obtained in the presence of gRNA for all sizes of homology arms (Figure 3c, left panel), suggesting that the plasmid-based gRNA/Cas9 system also is very efficient.Figure 3

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