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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

Multiple simultaneous integration of three β-carotene pathway genes. a Picture of colonies after simultaneous integration of BTS1, crtYB and crtI on plates without selection. S. cerevisiae (TC-3) harboring a centromeric plasmid constitutively expressing cas9 was co-transformed with: left empty vector control and linearized donor DNAs encoding BTS1, crtYB and crtI. Right gRNA vector expressing three gRNAs targeting intergenic loci XII-5, XI-2 and X-3, and linearized donor DNAs encoding BTS1, crtYB and crtI. Colonies with successful pathway integration accumulate β-carotene, resulting in an orange pigmentation. b Percentage of complete pathway integration with and without the expression of targeting gRNAs. c Frequency of correct simultaneous integration of the three genes BTS1, crtYB, and crtI at the specific intergenic loci XII-5, XI-2 and X-3, respectively, determined by genotyping. d HPLC analysis of β-carotene production of three independent orange colonies and a non-producing strain as control (CEN.PK113-7D). The experiment was repeated twice and error bars in all panels represent 95% confidence intervals.
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Fig4: Multiple simultaneous integration of three β-carotene pathway genes. a Picture of colonies after simultaneous integration of BTS1, crtYB and crtI on plates without selection. S. cerevisiae (TC-3) harboring a centromeric plasmid constitutively expressing cas9 was co-transformed with: left empty vector control and linearized donor DNAs encoding BTS1, crtYB and crtI. Right gRNA vector expressing three gRNAs targeting intergenic loci XII-5, XI-2 and X-3, and linearized donor DNAs encoding BTS1, crtYB and crtI. Colonies with successful pathway integration accumulate β-carotene, resulting in an orange pigmentation. b Percentage of complete pathway integration with and without the expression of targeting gRNAs. c Frequency of correct simultaneous integration of the three genes BTS1, crtYB, and crtI at the specific intergenic loci XII-5, XI-2 and X-3, respectively, determined by genotyping. d HPLC analysis of β-carotene production of three independent orange colonies and a non-producing strain as control (CEN.PK113-7D). The experiment was repeated twice and error bars in all panels represent 95% confidence intervals.

Mentions: Using the plasmid-based CrEdit system, cells expressing Cas9 were simultaneously transformed with three different large EasyClone donor DNAs for integration of PTDH3-crtI (6.6 kb), PTEF1-crtYB (5.8 kb), and PPGK1-BTS1 (5.1 kb) into three intergenic sites X-3, XI-2, and XII-5 situated on different chromosomes, using 500 bp homology arms. The cells were co-transformed with one episomal vector expressing the three gRNAs targeting these three sites, or with the empty vector for the −gRNA control. Transformants were plated on media selecting only for Cas9 and gRNA expressing plasmids. We observed that 84% of the derived colonies presented orange pigment formation when the gRNAs were present, indicating complete β-carotene pathway integration. In contrast, we only observed white colonies when the gRNAs were absent, indicating that no correct triple integration had been achieved (Figure 4a). All colonies were then replicated on single drop-out plates in order to screen for the integration of the three independent selection marker genes. As expected, all orange colonies were positive for all the three marker genes (Figure 4b, left panel). We subsequently tested the genotype of 32 orange colonies at the three expected integration sites, and observed 100% correct integration, thereby confirming complete pathway assembly (Figure 4b, right panel). In addition, we measured β-carotene levels by HPLC in three confirmed clones, and demonstrated that 12.7 ± 2.5 mg L−1 β-carotene was produced (Figure 4c). This proves the ability of the CrEdit system to simultaneously integrate three large DNA fragments with surprisingly high efficiency (84%) at the correct loci even without selection pressure. As for comparison, simultaneous integration of three genes has previously been demonstrated with 44% efficiency when relying on native HR alone and when applying selective pressure [12]. It was furthermore attempted to repeat the multi-loci pathway integration using short homology arms (60 bp) to investigate if we could simply use PCR products directly as donors for the multiplex integration. However, no viable colonies grew on the plates even after 1 week of incubation (data not shown), indicating that longer homology arms are beneficial for multiplex genome integrations. We assume this may due to the fact that multi-loci pathway integration is quite demanding with regards to coordinated repair activity, and long homology arms are easier to utilize for the native yeast HR machinery, thereby enabling correct simultaneous integration at multiple loci.Figure 4


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)

Multiple simultaneous integration of three β-carotene pathway genes. a Picture of colonies after simultaneous integration of BTS1, crtYB and crtI on plates without selection. S. cerevisiae (TC-3) harboring a centromeric plasmid constitutively expressing cas9 was co-transformed with: left empty vector control and linearized donor DNAs encoding BTS1, crtYB and crtI. Right gRNA vector expressing three gRNAs targeting intergenic loci XII-5, XI-2 and X-3, and linearized donor DNAs encoding BTS1, crtYB and crtI. Colonies with successful pathway integration accumulate β-carotene, resulting in an orange pigmentation. b Percentage of complete pathway integration with and without the expression of targeting gRNAs. c Frequency of correct simultaneous integration of the three genes BTS1, crtYB, and crtI at the specific intergenic loci XII-5, XI-2 and X-3, respectively, determined by genotyping. d HPLC analysis of β-carotene production of three independent orange colonies and a non-producing strain as control (CEN.PK113-7D). The experiment was repeated twice and error bars in all panels represent 95% confidence intervals.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492099&req=5

Fig4: Multiple simultaneous integration of three β-carotene pathway genes. a Picture of colonies after simultaneous integration of BTS1, crtYB and crtI on plates without selection. S. cerevisiae (TC-3) harboring a centromeric plasmid constitutively expressing cas9 was co-transformed with: left empty vector control and linearized donor DNAs encoding BTS1, crtYB and crtI. Right gRNA vector expressing three gRNAs targeting intergenic loci XII-5, XI-2 and X-3, and linearized donor DNAs encoding BTS1, crtYB and crtI. Colonies with successful pathway integration accumulate β-carotene, resulting in an orange pigmentation. b Percentage of complete pathway integration with and without the expression of targeting gRNAs. c Frequency of correct simultaneous integration of the three genes BTS1, crtYB, and crtI at the specific intergenic loci XII-5, XI-2 and X-3, respectively, determined by genotyping. d HPLC analysis of β-carotene production of three independent orange colonies and a non-producing strain as control (CEN.PK113-7D). The experiment was repeated twice and error bars in all panels represent 95% confidence intervals.
Mentions: Using the plasmid-based CrEdit system, cells expressing Cas9 were simultaneously transformed with three different large EasyClone donor DNAs for integration of PTDH3-crtI (6.6 kb), PTEF1-crtYB (5.8 kb), and PPGK1-BTS1 (5.1 kb) into three intergenic sites X-3, XI-2, and XII-5 situated on different chromosomes, using 500 bp homology arms. The cells were co-transformed with one episomal vector expressing the three gRNAs targeting these three sites, or with the empty vector for the −gRNA control. Transformants were plated on media selecting only for Cas9 and gRNA expressing plasmids. We observed that 84% of the derived colonies presented orange pigment formation when the gRNAs were present, indicating complete β-carotene pathway integration. In contrast, we only observed white colonies when the gRNAs were absent, indicating that no correct triple integration had been achieved (Figure 4a). All colonies were then replicated on single drop-out plates in order to screen for the integration of the three independent selection marker genes. As expected, all orange colonies were positive for all the three marker genes (Figure 4b, left panel). We subsequently tested the genotype of 32 orange colonies at the three expected integration sites, and observed 100% correct integration, thereby confirming complete pathway assembly (Figure 4b, right panel). In addition, we measured β-carotene levels by HPLC in three confirmed clones, and demonstrated that 12.7 ± 2.5 mg L−1 β-carotene was produced (Figure 4c). This proves the ability of the CrEdit system to simultaneously integrate three large DNA fragments with surprisingly high efficiency (84%) at the correct loci even without selection pressure. As for comparison, simultaneous integration of three genes has previously been demonstrated with 44% efficiency when relying on native HR alone and when applying selective pressure [12]. It was furthermore attempted to repeat the multi-loci pathway integration using short homology arms (60 bp) to investigate if we could simply use PCR products directly as donors for the multiplex integration. However, no viable colonies grew on the plates even after 1 week of incubation (data not shown), indicating that longer homology arms are beneficial for multiplex genome integrations. We assume this may due to the fact that multi-loci pathway integration is quite demanding with regards to coordinated repair activity, and long homology arms are easier to utilize for the native yeast HR machinery, thereby enabling correct simultaneous integration at multiple loci.Figure 4

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