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

Microbial Cell Factories

Research

Michael Krogh Jensen¹  Scott James Harrison¹  Jérôme Maury¹  Carlotta Ronda¹  Alex Toftgaard Nielsen¹  Jay D Keasling¹  Simo Abdessamad Baallal Jacobsen¹  Susanne Manuela Germann¹  Irina Borodina¹  Tadas Jakočiu̅nas¹ 

[1]The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark

关键词: Metabolic engineering;    CRISPR/Cas9;    Genome editing;    Saccharomyces cerevisiae;    Carotenoid production;    Genome integrations;   

DOI  :  10.1186/s12934-015-0288-3

 received in 2015-02-08, accepted in 2015-06-22,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundOne 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.ResultsHere, 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© Ronda et al. 2015