Genome Engineering of Filamentous Fungi

1. Service Overview

Filamentous fungi genome modification services provide targeted genetic engineering solutions for both common standard filamentous fungal strains (e.g., Trichoderma reesei, Aspergillus niger, Penicillium rubens, Aspergillus oryzae, Aspergillus terreus) and wild-type filamentous fungal strains. These services cover a comprehensive range of genetic editing types, including single-gene knockout, multi-gene knockout, gene knock-in, and base editing (cytosine base editing/CBE and adenine base editing/ABE). The core objectives of the services are to support research on fungal gene function, optimize the performance of industrial strains (e.g., enhancing the yield of enzymes or organic acids), and facilitate the development of pharmaceutical-related metabolites (e.g., statins, antibiotics). For standard strains, the project cycle is 3–6 months, while wild-type strains require customized evaluation to determine the specific timeline.

2. Technical Principles

The genome modification of filamentous fungi primarily relies on the CRISPR/Cas9 system, following a four-step technical process:

2.1 Target Design

（1）Determine target genes: Select genes to be edited based on research objectives, such as genes associated with secondary metabolite synthesis or drug resistance.

（2）sgRNA design: Use specialized software (e.g., E-CRISP, CHOPCHOP, Cas-OFFinder) to design single-guide RNAs (sgRNAs) targeting the target genes. This ensures high sgRNA specificity to minimize off-target effects. sgRNAs need to recognize a 20-nucleotide sequence upstream of the protospacer adjacent motif (PAM) site; for the Cas9 system, the common PAM sequence is 5′-NGG-3′.

2.2 Preparation of Editing Components

（1）Construction of Cas9 protein expression vector: Optimize the Cas9 gene sequence according to the codon preference of the target filamentous fungus, and add nuclear localization signals (NLS) to both ends of the sequence to construct a Cas9 protein expression vector.

（2）Construction of sgRNA expression vector: The expression of sgRNA requires driving by a promoter; commonly used promoters include RNA polymerase III-type U6 promoter, tRNA promoter, and 5S rRNA promoter.

2.3 Delivery of Editing Components

（1）Plasmid transformation: Introduce the constructed Cas9 expression vector and sgRNA expression vector into filamentous fungal cells via plasmid transformation. Common transformation methods include protoplast transformation and Agrobacterium-mediated transformation.

（2）Autonomously replicating plasmids: Construct autonomously replicating plasmids using the AMA1 sequence from
Aspergillus nidulans. These plasmids can replicate autonomously outside the chromosome of filamentous fungi, improving transformation efficiency. Additionally, the plasmids can be removed through subculture under non-selective conditions, enabling cyclic use of selection markers.

（3）Ribonucleoprotein (RNP) complex delivery: Combine in vitro transcribed sgRNA with purified Cas9 protein to form an RNP complex, which is then delivered into filamentous fungal cells via microinjection or electroporation. This delivery method eliminates the need for expression cassette optimization, reduces construction steps, and the transient existence of RNP in vivo lowers off-target risks.

2.4 Screening and Verification of Editing Efficiency

（1）Selection marker screening: Use selection markers such as antibiotic resistance genes or auxotrophic genes to screen transformants that have successfully incorporated the editing components.

（2）Molecular biology verification: Verify transformants through PCR and DNA sequencing to detect whether the target gene has been correctly edited (e.g., gene knockout, knock-in, or base editing).

3. Technical Flowchart

Fig.1 CRISPR-based genome modification strategies for filamentous fungi

4. Technical Features

4.1 High Efficiency

（1）High editing efficiency: The CRISPR/Cas system achieves a single-gene editing efficiency of 93%–100% in filamentous fungi, significantly higher than traditional genetic engineering methods.

（2）Multi-gene editing capability: It can simultaneously edit multiple genes to achieve co-knockout or co-expression of multiple genes, facilitating the study of complex metabolic pathways and regulatory networks.

4.2 High Precision

（1）Strong targeting: sgRNA can accurately recognize the target gene sequence and guide Cas9 nuclease to cleave DNA at specific sites, enabling precise genetic editing.

（2）Precise editing via HR repair pathway: The homologous recombination (HR) repair pathway supports precise operations such as gene knock-in and replacement, ensuring high editing accuracy.

4.3 Flexibility

（1）Diverse editing methods: It can realize multiple editing modes, including gene knockout, knock-in, base editing, and expression regulation, to meet different research needs.

（2）Customizable components: sgRNA design, promoter selection, and delivery methods can be customized according to different filamentous fungal species and research objectives.

4.4 Wide Application Range

It is applicable to basic research (e.g., gene function, metabolic pathway analysis), industrial strain improvement (e.g., enhancing enzyme production), and pharmaceutical development (e.g., analyzing natural product biosynthesis pathways).

5. Application Scenarios

5.1 Pharmaceutical Field

Filamentous fungi are important producers of pharmaceutical ingredients. They can synthesize various antibiotics (e.g., penicillin, cephalosporins), immunosuppressants (e.g., cyclosporine, mycophenolic acid), and lipid-lowering drugs (e.g., statins produced by
A. terreus), providing critical pharmaceutical resources for human health.

5.2 Industrial Production

As the main producers of industrial enzymes, filamentous fungi produce enzymes (e.g., cellulases from T. reesei, glucoamylases and pectinases from A. niger) that are widely used in the food, chemical, and textile industries. Additionally, they can be used to produce organic acids (e.g., citric acid from A. niger) and biofuels.

5.3 Agricultural Field

Some filamentous fungi can be used for biological control to inhibit the growth of plant pathogens. They also play an important role in the degradation of agricultural waste and soil improvement.

5.4 Basic Biological Research

Filamentous fungi (e.g., A. oryzae) serve as model organisms for studying gene function, metabolic pathways, and cell differentiation, providing an important model for life science research.

6. Delivery Content and Standards

（1）PCR and DNA sequencing verification data of target sites (to confirm the correctness of gene editing).

（2）Two glycerol stocks of the engineered strain (for long-term preservation and subsequent experiments).

（3）mRNA transcription level data (provided specifically for overexpression projects, to verify the expression efficiency of target genes).

（4）Project final report (including detailed experimental procedures, results analysis, and data summaries).

7. References

[1] Shi TQ, Gao J, Wang WJ, et al. CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production.ACS Synth Biol. 2019;8(2):445-454. doi:10.1021/acssynbio.8b00478

[2] Jiang C, Lv G, Tu Y, et al. Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi. Front Microbiol. 2021;12:638096. Published 2021 Feb 11. doi:10.3389/fmicb.2021.638096

[3] Huang L, Dong H, Zheng J, Wang B, Pan L. Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion.Microbiol Res. 2019;223-225:44-50. doi:10.1016/j.micres.2019.03.007