Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
Open accessAbstract
Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.
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A multifunctional CRISPR system performs dual and tri-functional base editing but suffers from the requirement of each Cas protein specific to its own PAM sequence.
“It provides a multifunctional CRISPR system that performs dual and tri-functional base editing but suffers from the requirement of each Cas protein specific to its own PAM sequence (Lian et al., 2017).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Lian et al., 2017
The Discussion (page unavailable) describes dual-functional CRISPR systems and the paper’s tri-functional CRISPR-AID system, which uses proteins with different PAM sequences. But its functions are transcriptional activation, transcriptional interference, and gene deletion—not dual- or tri-functional base editing. The passage describes distinct PAM sequences as part of the system’s design, not explicitly as a drawback. (LLM, conf=0.86)
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“It provides a multifunctional CRISPR system that performs dual and tri-functional base editing but suffers from the requirement of each Cas protein speci fi c to its own PAM sequence (Lian et al., 2017).”
- Authors
- Lian, J., HamediRad, M., Hu, S., & Zhao, H.
- Affiliations
- University of Illinois Urbana-Champaign (US)
- Corresponding author
- Zhao, Huimin
- Venue
- Nature Communications
- Published
- 2017-11-22
- Volume
- 8
- Issue
- 1
- Pages
- 1688-1688
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC5700065
- OpenAlex
- W2769160949
- Cited by
- 319
- References
- 56
- Citations by year
- 2026: 21 · 2025: 28 · 2024: 33 · 2023: 34 · 2022: 42 · 2021: 51 · 2020: 42 · 2019: 44 · 2018: 21 · 2017: 1
- FWCI
- 12.06
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- U.S. Department of Energy (DESC0018260)
- University of Illinois at Urbana-Champaign
- Carl R. Woese Institute for Genomic Biology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- Saccharomyces cerevisiae, transcriptional activation, transcriptional interference, gene deletion, β-carotene production, yeast cell factories
- MeSH terms
- Cell Membrane, Cellulase, Genes, Fungal, Saccharomyces cerevisiae, Transcriptional Activation, Gene Deletion, beta Carotene, Saccharomyces cerevisiae Proteins, RNA Interference, Metabolic Networks and Pathways, Metabolic Engineering, CRISPR-Cas Systems
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