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A CRISPR/Cas9-based visual toolkit enabling multiplex integration at specific genomic loci in Aspergillus niger
Yangyang Li1,2,3, Cen Li4, Yishan Fu1,2,3
1National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, 214122, China.
Synthetic and Systems Biotechnology
|February 22, 2024
Summary
This study introduces a CRISPR-based multiplex integration toolkit for Aspergillus niger, enabling simultaneous gene insertion into multiple genomic sites. This accelerates the development of improved fungal cell factories for industrial applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Industrial Microbiology
Background:
- Aspergillus niger is a key fungus for industrial production, where enhanced recombinant gene expression is crucial.
- Increasing gene copy number boosts expression, but lengthy gene editing cycles in A. niger hinder rapid development.
- A "one-step" strategy for simultaneous integration into multiple genomic loci is needed to overcome these limitations.
Purpose of the Study:
- To develop a clustered regularly interspaced short palindromic repeats (CRISPR)-based multiplex integration toolkit for Aspergillus niger.
- To establish a "one-step" gene editing strategy for simultaneous integration of recombinant genes into multiple genomic loci.
- To enhance recombinant gene expression and accelerate the generation of improved A. niger cell factories.
Main Methods:
- A CRISPR-Cas9 homology-directed repair (CRISPR-HDR) system was constructed using CRISPR-Cas9 plasmid and hybrid donor DNAs (dDNAs).
- Optimization of homology arm length in dDNAs achieved 100% editing efficiency at target loci (bgl, amyA, ammA).
- A visual multigene editing system (VMS) with a sgRNA-tRNA array enabled concurrent double-strand breaks at multiple loci for multiplex integration.
Main Results:
- The toolkit achieved simultaneous replacement of three endogenous genes (bgl, amyA, ammA) with the xynA gene in 23.5% of white colonies.
- Simultaneous insertion of xynA into three loci resulted in a 48.6% increase in xynA activity compared to single-locus insertion.
- Enhanced expression of endogenous pelA and CALB was observed, demonstrating the toolkit's efficacy in boosting target gene expression.
Conclusions:
- The combined VMS and CRISPR-HDR system provides an efficient "one-step" strategy for multiplex gene integration in Aspergillus niger.
- This toolkit significantly accelerates the development of A. niger cell factories with enhanced production capabilities.
- The strategy facilitates rapid generation of improved fungal strains for diverse industrial applications.
