Abstract
Despite the advances made in genetic engineering of Saccharomyces cerevisiae, the multicopy genomic integration of large biochemical pathways remains a challenge. Here, we developed a Di-CRISPR (delta integration CRISPR-Cas) platform based on cleavage of the delta sites by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas) to enable unprecedented high-efficiency, multicopy, markerless integrations of large biochemical pathways into the S. cerevisiae genome. Detailed protocols are provided on the entire workflow which includes pDi-CRISPR plasmid and donor DNA construction, Di-CRISPR-mediated integration and analysis of integration efficiencies and copy numbers through flow cytometry and quantitative polymerase chain reaction (qPCR).
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Acknowledgments
We thank Agency for Science, Technology, and Research, Singapore for supporting various research projects in the Metabolic Engineering Research Laboratory (MERL) through the Visiting Investigator Programme to H.Z (1535j00137).
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Shi, S., Liang, Y., Ang, E.L., Zhao, H. (2019). Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae. In: Santos, C., Ajikumar, P. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 1927. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9142-6_6
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DOI: https://doi.org/10.1007/978-1-4939-9142-6_6
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