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Updated: Jun 1, 2025
09:04
Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
8.2K
Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.
Senqin Hou1,2,3, Shibin Yang1,2, Wenqin Bai4,5
1National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Microbial Cell Factories
|January 21, 2025
Summary
A new CRISPR-Cpf1 system significantly improves genome editing in Ogataea polymorpha, enabling efficient multi-gene editing and enhancing its potential as an industrial cell factory.
Area of Science:
- Microbiology
- Molecular Biology
- Synthetic Biology
Background:
- Ogataea polymorpha is a methylotrophic yeast with potential for producing valuable compounds.
- Efficient genome editing tools are crucial for developing O. polymorpha into a cell factory.
- Existing CRISPR-Cas9 systems in O. polymorpha require improvement for multi-gene editing.
Purpose of the Study:
- To develop an efficient CRISPR-Cpf1-mediated genome editing system for O. polymorpha.
- To enhance homologous recombination (HR) efficiency for precise genome editing.
- To enable multiplexed gene deletion and integration for metabolic engineering.
Main Methods:
- Development of a CRISPR-Cpf1 system for O. polymorpha.
- Knockout of non-homologous end joining (NHEJ) related genes to boost HR.
- Application of the system for single, duplex, and triplex gene editing, including large DNA fragment manipulation.
Main Results:
- Achieved high editing efficiency for single (98.1%), duplex (93.9%), and triplex (94.0%) genes.
- Increased HR efficiency from <30% to 90-100% by knocking out NHEJ genes.
- Demonstrated >90% integration efficiency for triplex genes and >90% deletion of 20 kb fragments.
- Successfully produced lycopene in O. polymorpha via one-step integration of triple genes.
Conclusions:
- The CRISPR-Cpf1 system offers a powerful tool for multiplexed genome editing in O. polymorpha.
- This system facilitates efficient deletion and integration of multiple genes, accelerating metabolic engineering.
- The enhanced genome editing capabilities promise advancements in developing O. polymorpha as an industrial cell factory.
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