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Efficient multiplex CRISPR/Cpf1 (Cas12a) genome editing system in Aspergillus aculeatus TBRC 277
Dede Abdulrachman1, Verawat Champreda2, Lily Eurwilaichitr3
1Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand.
Journal of Biotechnology
|July 5, 2022
Summary
This study introduces a CRISPR/Cpf1 system for genome engineering in Aspergillus aculeatus, a filamentous fungus. The system, particularly using FnCpf1, enables efficient gene editing for industrial applications and strain improvement.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas technology is a powerful genome engineering tool.
- Cpf1 (Class 2, Type V) offers distinct features compared to Cas9.
- Its application in filamentous fungi remains limited.
Purpose of the Study:
- To develop and evaluate a single CRISPR/Cpf1 plasmid system for Aspergillus aculeatus.
- To assess the efficiency of different Cpf1 orthologs for genome editing.
- To demonstrate the system's utility for strain improvement in industrial biotechnology.
Main Methods:
- Tested three Cpf1 orthologs (AsCpf1, FnCpf1, LbCpf1) for site-specific DNA cleavage at the pksP locus.
- Investigated gene deletion of pksP and pyrG using a single crRNA array.
- Performed simultaneous multi-site editing and targeted disruption of the NHEJ pathway.
Main Results:
- FnCpf1 exhibited the highest editing efficiency (93%) among tested orthologs.
- Successful deletion of 1.7 kb and 0.5 kb fragments from pksP and pyrG genes.
- Achieved 40% combined editing efficiency for three genomic sites, including kusA.
- Disruption of the NHEJ pathway enhanced gene targeting precision and efficiency.
Conclusions:
- The developed CRISPR/Cpf1 system is effective for genome engineering in Aspergillus aculeatus.
- This technology facilitates precise gene editing and strain improvement for industrial bioproduct production.
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