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A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment
Zhen Chen1, Wei Hong1, Xiaorong Wei1
1State Key Laboratory of Bioreactor Engineering, Qingdao Innovation Institute of East China University of Science and Technology, East China University of Science and Technology, Shanghai 200237, China.
ACS Synthetic Biology
|April 29, 2026
Summary
A new CRISPR/Cas9 system enables multiplex genome editing in Acremonium chrysogenum, improving cephalosporin C production. This tool facilitates gene function studies and metabolic engineering for antibiotic development.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Cephalosporin C (CPC) antibiotics are vital, with Acremonium chrysogenum as the sole industrial producer.
- Limited multiplex genome-editing tools hinder A. chrysogenum gene function and metabolic engineering studies.
Purpose of the Study:
- Develop a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system for A. chrysogenum.
- Enable one-step multilocus knockout, DNA deletion, and gene overexpression.
Main Methods:
- A CRISPR/Cas9 system utilizing endogenous tRNA promoters for multiplex genome editing was developed.
- Heterologous mCherry protein expression was used to introduce a visible red phenotype.
- Endogenous tRNA promoters were evaluated for knockout efficiency, with tRNAVal showing the highest.
Main Results:
- The system achieved efficient double- and triple-site knockouts in an industrial A. chrysogenum strain.
- A 50.7-kb DNA fragment (sorbicillinoids gene cluster) was deleted with nearly 100% efficiency.
- Overexpression of pcbAB increased CPC titer by 23.8% (from 5.59 g/L to 6.92 g/L).
Conclusions:
- The developed tRNA-gRNA array-based CRISPR/Cas9 system is a versatile platform for A. chrysogenum functional genomics.
- This system significantly advances industrial strain engineering for improved antibiotic production.
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