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A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae
Yueping Zhang1, Juan Wang1, Zibai Wang1
1Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 100029, Beijing, China.
Nature Communications
|March 7, 2019
Summary
We developed GTR-CRISPR and Lightning GTR-CRISPR systems for efficient multiplexed gene editing in Saccharomyces cerevisiae. These tools accelerate strain engineering for synthetic biology applications, enabling rapid genetic modifications and pathway optimization.
Area of Science:
- Synthetic Biology
- Molecular Biology
- Biotechnology
Background:
- Strain engineering is a bottleneck in synthetic biology due to limitations in multiplexed gene editing.
- CRISPR-Cas9 technology offers precise genome modification capabilities.
Purpose of the Study:
- To develop a novel CRISPR-Cas9 system for multiplexed gene editing in Saccharomyces cerevisiae.
- To accelerate the strain engineering process for synthetic biology applications.
Main Methods:
- Developed GTR-CRISPR (gRNA-tRNA array for CRISPR-Cas9) for simultaneous gene disruption.
- Created Lightning GTR-CRISPR, an accelerated system avoiding cloning steps.
- Applied Lightning GTR-CRISPR for yeast lipid network simplification.
Main Results:
- Achieved 8-gene disruption with 87% efficiency using GTR-CRISPR.
- Enabled 6-gene disruption in 3 days with 60% efficiency using Lightning GTR-CRISPR.
- Increased free fatty acid production by 30-fold in 10 days via targeted gene deletions.
Conclusions:
- GTR-CRISPR and Lightning GTR-CRISPR significantly enhance multiplexed strain engineering in yeast.
- These systems accelerate synthetic biology workflows and enable efficient metabolic engineering.
- The developed tools are valuable for advancing synthetic biology and automation.
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