title: A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.
abstract: Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.
authors.affiliation: Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Shuobo
authors.initials: S
authors.lastname: Shi
authors.affiliation: Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Youyun
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Mingzi M
authors.initials: MM
authors.lastname: Zhang
authors.affiliation: Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore. Electronic address: angel@merl.a-star.edu.sg.
authors.forename: Ee Lui
authors.initials: EL
authors.lastname: Ang
authors.affiliation: Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore, Singapore; Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States. Electronic address: zhao5@illinois.edu.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D015825:Chromosomes, Fungal / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins
keywords: CRISPR-Cas; Delta integration; Genome editing; Multi-copy integration; Saccharomyces cerevisiae