Aureobasidium melanogenum is an abundant yeast species inhabiting mangrove ecosystems. Beyond its considerable application potential, this strain plays important ecological roles in maintaining mangrove ecosystem balance and facilitating biogeochemical cycling, making it an important research target in marine microbiology. However, the genetic background of A. melanogenum remains poorly characterized, and the lack of efficient gene editing tools has greatly limited its functional studies and engineering applications. To address this challenge, we designed a CRISPR-Cas9-based knockout system—CRISPR-Cas9-NAT vector—for A. melanogenum, providing a platform for subsequent functional validation of key genes and strain engineering.
To enable efficient genome editing, we built upon existing fungal CRISPR-Cas9 systems. First, the plasmid p414-TEF1p-Cas9-CYC1t (Addgene #43802) was retrieved, and restriction enzyme site analysis identified SwaI and KpnI as suitable double digestion sites for linearization. Next, the nat gene (encoding nourseothricin resistance, NAT) was amplified from the laboratory-preserved fl4a-nat-loxp plasmid using primers designed according to seamless cloning principles, with primer R introducing a PacI site. This fragment was inserted into the linearized backbone, generating the Cas9-NAT plasmid.
Subsequently, PacI digestion was employed to introduce the AMA1 autonomous replication sequence from Aspergillus nidulans. The AMA1 fragment, amplified with NotI-flanked primers, was seamlessly cloned into the Cas9-NAT plasmid, yielding Cas9-NAT-AMA1. Finally, by NotI digestion of the Cas9-NAT-AMA1 plasmid, a synthetic U6 promoter–sgRNA–scaffold–U6 terminator cassette was introduced via seamless cloning, generating the final CRISPR-Cas9-Am knockout vector. This modular system allows simple replacement of the sgRNA region to enable targeted editing of diverse genes in A. melanogenum.
The melanin-producing yeast Aureobasidium melanogenum is a common and ecologically important taxon in typical marine environments such as mangroves, where it plays a crucial role in biogeochemical cycling and ecosystem stability. Owing to its remarkable metabolic diversity, it also exhibits promising potential for biotechnological applications (Masi et al., 2024). However, compared with model fungi, genetic studies on this group remain very limited, and the absence of mature molecular tools and gene manipulation systems has constrained its in-depth investigation. At present, gene knockout in A. melanogenum primarily relies on homologous recombination, but the method suffers from low positive rates and unsatisfactory efficiency, making it difficult to support systematic functional genomics research.
Meanwhile, CRISPR-Cas9 technology has emerged as the most widely used gene editing strategy in eukaryotic microorganisms. In model fungi such as Saccharomyces cerevisiae and certain filamentous fungi, this system has been thoroughly validated and successfully applied to both metabolic engineering and functional genomics (DiCarlo et al., 2013; Nødvig et al., 2015). However, studies in A. melanogenum and other non-model fungi are still scarce, and there is a clear lack of efficient and broadly applicable editing tools. Considering the limitations of the homologous recombination system currently available in our laboratory, the development of a high-efficiency CRISPR-Cas9-Am system would markedly enhance gene editing capacity and expand the prospects for molecular genetics and synthetic biology applications in this species.
During the construction of the CRISPR-Cas9-Am system, careful consideration was given to the choice of each vector component to ensure efficient and stable gene editing in the fungal host.
The NAT (Nourseothricin resistance gene) was selected as the selectable marker. Unlike other commonly used resistance markers such as ampicillin or hygromycin, NAT has been shown to exhibit low background resistance in fungal cells, while providing high screening efficiency. Its applicability has been validated in a wide range of non-model fungi (Krappmann, 2007). Therefore, NAT was chosen to ensure accurate and universal selection in Aureobasidium melanogenum.
The AMA1 (Autonomous Maintenance in Aspergillus 1) element, originally identified in Aspergillus flavus, was incorporated as an autonomous replication sequence. AMA1 has been demonstrated to maintain plasmids at high copy numbers and ensure stable maintenance across a variety of filamentous fungi and yeasts (Masi et al., 2024). In contrast, the Saccharomyces cerevisiae ARS (Autonomously Replicating Sequence) functions efficiently only in S. cerevisiae and exhibits limited compatibility with other fungi (DiCarlo et al., 2013). To improve plasmid stability and transformation efficiency in A. melanogenum, AMA1 was selected instead of ARS.
The TEF (Translational Elongation Factor 1α) promoter was employed as a strong constitutive promoter, given its ability to drive robust and stable expression of exogenous genes in fungi (Jakočiūnas et al., 2015). Compared to mammalian-derived promoters such as CMV, the TEF promoter offers superior transcriptional recognition in fungal hosts, thereby ensuring sustained and efficient Cas9 expression to maximize editing efficiency.
The CYC1 terminator, derived from the S. cerevisiae cytochrome c1 gene, was used as a transcriptional termination signal. It is one of the most widely applied terminators in fungal expression systems and has been shown to ensure accurate transcriptional termination while enhancing mRNA stability (DiCarlo et al., 2013). Its incorporation into the Cas9 expression cassette enhances the reliability of Cas9 production.
For sgRNA expression, the U6 promoter and U6 terminator were employed. As an RNA polymerase III promoter, U6 is widely used to drive the transcription of short RNAs, including sgRNAs, with precise 5′ ends, making it particularly suitable for CRISPR systems (Ran et al., 2013). Both endogenous and heterologous U6 promoters have been successfully applied in fungal CRISPR platforms to drive high-level sgRNA expression (Zheng et al., 2018). Accordingly, in this study, the U6 promoter and terminator were adopted to ensure efficient sgRNA expression and functionality in A. melanogenum.
The plasmid map of p414-TEF1p-Cas9-CYC1t was obtained from Addgene, and its restriction enzyme sites were analyzed.
Based on the analysis, SwaI and KpnI were selected as restriction sites to linearize the plasmid.
Using the laboratory-constructed plasmid fl4a-nat-loxp as a template, primers were designed based on seamless cloning principles to amplify the nourseothricin resistance gene (NAT). The reverse primer was engineered to introduce a PacI restriction site. The corresponding primer sequences are listed below.
Using the principles of seamless cloning, the amplified PGK-NAT-PolyA fragment was inserted into the linearized p414-TEF1p-Cas9-CYC1t plasmid, resulting in the construction of the Cas9-NAT plasmid vector, as shown below.
Restriction site analysis of the Cas9-NAT plasmid was performed, and PacI was selected as the restriction site for linearization.
Using the Aspergillus flavus genome as a template, primers AMA1-F/AMA1-R were designed to PCR-amplify the AMA autonomously replicating sequence, with the AMA1-R primer incorporating a NotI restriction site. The amplified fragment was then seamlessly cloned into the Cas9-NAT vector to construct the Cas9-NAT-AMA1 plasmid. The plasmid map and primer design are shown below.
The gRNA expression fragment, containing the U6 promoter, sgRNA, gRNA scaffold, and U6 terminator, was synthesized. Primers U6T-F and U6P-R were designed to PCR-amplify the fragment comprising the U6 Terminator + sgRNA + gRNA scaffold + U6 Promoter. The map of the gRNA expression fragment is shown below.
The Cas9-NAT-AMA plasmid was linearized using NotI restriction enzyme, and the above fragment was inserted into the linearized plasmid via seamless cloning to obtain the final CRISPR-Cas9-Am knockout vector. In practical applications, the sgRNA can be replaced with the sgRNA targeting the gene of interest. The map of the CRISPR-Cas9-Am knockout vector is shown below.
The p414-TEF1p-Cas9-CYC1t plasmid was used as the backbone and double-digested with SwaI and KpnI (37 °C, 4 h), followed by recovery of the 7087 bp fragment.
Using fl4a-nat-loxp as the template, primers NAT-oF/NAT-oR were designed to PCR-amplify the NAT expression cassette. The PCR product was recovered from agarose gel and inserted into the linearized vector via a seamless cloning system (37 °C, 30 min).
The ligation product was transformed into E. coli, and positive colonies were screened by colony PCR. After verification by gel electrophoresis, the correct transformants were cultured in LB medium containing ampicillin, and the plasmids were extracted and confirmed by sequencing.
The Cas9-NAT plasmid was digested with PacI to obtain the vector backbone.
Using the Aspergillus flavus genome as a template, primers AMA-F/AMA-R were designed to PCR-amplify the AMA autonomously replicating sequence, with a NotI restriction site added to the AMA-F primer.
The AMA fragment was inserted into the vector via seamless cloning to construct the Cas9-NAT-AMA plasmid. The plasmid was transformed into E. coli, and the correct clone was confirmed by sequencing.
The gRNA expression fragment, containing the U6 promoter, gRNA scaffold, and U6 terminator, was designed and synthesized by a commercial company. Primers U6T-F and U6P-R were designed to amplify the fragment comprising the U6 promoter and terminator + gRNA scaffold.
The PCR product was directly recovered using a DNA purification kit and seamlessly cloned into the NotI-linearized Cas9-NAT-AMA1 plasmid.
The resulting plasmid was transformed into E. coli, and positive clones were screened by PCR and confirmed by sequencing. The final construct was the CRISPR-Cas9-Am knockout vector.
The target gene CDS sequence was input into the CRISPR online design tool (https://crispor.gi.ucsc.edu/) to select sgRNA sequences with high specificity and low off-target risk.
The selected sgRNA sequence can then be inserted into the vector to achieve site-specific editing of the target gene.
In the construction and application of the CRISPR-Cas9-Am system, validating its feasibility and editing efficiency in host cells is a crucial step. To directly and accurately assess the functionality of the constructed CRISPR-Cas9-Am knockout vector, the Ade2 gene was selected as a target for verification.
The Ade2 gene encodes phosphoribosyl pyrophosphate amidotransferase (PRPP amidotransferase), a key enzyme in the de novo purine biosynthesis pathway. Deletion of Ade2 blocks adenine synthesis, leading to the accumulation of metabolic intermediates that are oxidized within the cell to form a red compound, resulting in colonies with a distinct red phenotype. This phenotypic change is intuitive, stable, and easy to observe, making it an ideal indicator for verifying the functionality of the CRISPR-Cas9 knockout system. Therefore, by targeting Ade2 and observing changes in colony color, the effectiveness of the constructed CRISPR-Cas9-Am vector in gene editing can be rapidly assessed.
Plasmid vectors: p414-TEF1p-Cas9-CYC1t, fl4a-nat-loxp
Strains: Escherichia coli DH5α, Aureobasidium melanogenum P16
Other reagents: Restriction enzymes SwaI, KpnI, NotI, PacI; seamless cloning kit; DNA extraction and gel purification kits; nourseothricin.
PCR thermal cycler, electrophoresis apparatus with gel imaging system, biosafety cabinet, shaker, electroporator, centrifuge, etc.
The p414-TEF1p-Cas9-CYC1t plasmid was double-digested with SwaI and KpnI at 37 °C for 4 h, and the linearized fragment was recovered. Using fl4a-nat-loxp as the template, the NAT fragment was PCR-amplified and inserted into the linearized vector via seamless cloning. The ligation product was transformed into E. coli, and positive clones were confirmed by PCR and sequencing, yielding the Cas9-NAT plasmid.
The AMA fragment was amplified from the Aspergillus flavus genome and inserted into the PacI-digested Cas9-NAT plasmid via seamless cloning. The construct was transformed and verified by sequencing, resulting in the Cas9-NAT-AMA plasmid.
Using a commercially synthesized plasmid containing the gRNA expression fragment as a template, the U6 promoter, terminator, and gRNA scaffold were PCR-amplified and seamlessly cloned into the NotI-digested Cas9-NAT-AMA plasmid to construct the complete CRISPR-Cas9-Am vector.
The CRISPR online design tool (crispor) was used to select sgRNA sequences targeting the Ade2 gene with high specificity and minimal off-target effects. The selected sgRNA was inserted into the vector. Protoplast transformation was performed, followed by primary screening on YPD plates containing nourseothricin (50% resistance) and secondary screening (100% resistance). Positive colonies were sequenced for verification.
The constructed CRISPR-Cas9-Am-ΔAde2 vector was transformed into the P16 strain, and nourseothricin-resistant transformants were selected. Deletion of the Ade2 gene was confirmed by PCR and sequencing. Colony color was observed: successful deletion of Ade2 resulted in red colonies due to disrupted adenine metabolism.
Using the CRISPOR website, the CDS sequence of the Ade2 gene from the P16 strain was entered into the Step 1 Input box. In Step 2, the Aureobasidium pullulans genome data was selected, and in Step 3, the option 20 bp-NGG-SpCas9 was chosen, followed by submission.
The predicted optimal sgRNA parameters are summarized in the table below.
The reverse complement of the above sgRNA sequence was inserted into the plasmid vector. The reverse complement sequence is shown below:
5’-AGTCAACATTCTGGACGCAG-3’
The NAT selectable marker expression cassette was successfully inserted, as verified by primer design and colony PCR followed by gel electrophoresis.
The AMA1 autonomous replicating sequence was successfully inserted, as confirmed by primer design and colony PCR with gel electrophoresis.
The U6 promoter, sgRNA, and gRNA scaffold expression cassette was successfully inserted, verified by primer design and colony PCR with gel electrophoresis.
Plasmids with correct sequences were transformed into Aureobasidium melanogenum P16 via protoplast transformation. Cultures were grown at 28 °C to observe growth, and secondary screening results are shown in the figure. A clear color difference was observed between Ade2 knockout and wild-type colonies. Deletion of the Ade2 gene blocks adenine biosynthesis, leading to accumulation of red metabolic intermediates and resulting in a red colony phenotype. The experiment showed a knockout efficiency of approximately 50%, demonstrating the successful construction of the CRISPR-Cas9-Am knockout vector, which can be used for gene deletion in A. melanogenum P16.
By targeting the Ade2 gene, this study verified the feasibility of the CRISPR-Cas9-Am vector for genome editing in Aureobasidium melanogenum P16. The colony color change resulting from Ade2 deletion provided a clear and reliable phenotypic marker, confirming that the vector enables efficient site-specific gene knockout. Subsequent applications of this system in the laboratory showed a positive rate of 50%–80% for gene editing.
This study employed the DBTL (Design–Build–Test–Learn) engineering cycle to gradually establish a CRISPR-Cas9-Am gene editing system suitable for Aureobasidium melanogenum P16. Through two complete rounds of the DBTL cycle, we achieved a transition from initial design failures to the successful construction of the system.
In the first design phase, we referred to experiences from model fungi and yeast, selecting the following elements:
ARS replication element: The ARS (Autonomously Replicating Sequence) is widely used in Saccharomyces cerevisiae for plasmid maintenance. It is structurally simple and well-characterized, making it a suitable choice for the plasmid replication origin (DiCarlo et al., 2013).
gpdA promoter: Derived from Aspergillus species, the gpdA promoter is a commonly used strong promoter reported to drive stable expression in various fungi (Meyer et al., 2011), and was therefore chosen to drive Cas9 expression.
NAT selectable marker: The nourseothricin resistance gene (NAT) exhibits low background resistance in fungi and has been validated as an effective selection marker (Krappmann, 2007).
CYC1 terminator and U6 promoter/terminator: Used for the Cas9 expression cassette and sgRNA expression cassette, respectively, both are common and reliable configurations (Ran et al., 2013; Zheng et al., 2018).
Based on the above design, the first-generation CRISPR-Cas9 vector was constructed via seamless cloning and transformed into Aureobasidium melanogenum P16 for subsequent testing.
Testing revealed that sequencing showed extremely low copy numbers of the ARS sequence, indicating the plasmid was not stably maintained in P16. Cas9 expression driven by the gpdA promoter was insufficient, resulting in very low target gene knockout efficiency. Overall editing rates were far below expectations, with numerous false-positive transformants observed.
After the first cycle failure, we reviewed the literature and reflected on possible causes:
Limitations of ARS: ARS showed poor compatibility in non-model fungi and could not maintain stable plasmid replication.
Insufficiency of gpdA: Although effective in some fungi, it did not drive strong enough Cas9 expression in P16.
Improvement strategy: Based on literature, we decided to replace ARS with AMA1, which has been shown to efficiently maintain plasmids in various fungi (Masi et al., 2024), and to replace gpdA with the TEF1 promoter to enhance Cas9 expression (Jakočiūnas et al., 2015). The CYC1 terminator was retained as a high-efficiency terminator.
Based on reflections from the first cycle, we redesigned the second-generation vector.
Replication element: Replaced ARS with AMA1 to enhance plasmid stability in non-model fungi.
Cas9 promoter: Replaced gpdA with the TEF1 promoter to ensure high Cas9 expression.
Cas9 terminator: Retained CYC1 to improve transcription termination efficiency and mRNA stability.
NAT selectable marker and U6 framework: Remained unchanged, as they had already been validated.
Following the new design, the CRISPR-Cas9-Am system vector was reassembled and transformed into the P16 strain.
The improved system demonstrated significant performance enhancements. The AMA1 replication element successfully maintained the plasmid in P16, and sequencing indicated a notable increase in copy number. The TEF1 promoter drove high-level Cas9 expression, significantly improving gene knockout efficiency. In experiments targeting Ade2, knockout efficiency reached 50%–80%, confirming the feasibility and stability of the system.
The success of the second cycle indicated that AMA1 is a superior autonomous replicating sequence in non-model fungi, substantially enhancing plasmid stability. The TEF1 promoter outperforms gpdA in driving Cas9, demonstrating cross-species expression capability. The choices of CYC1 and U6 were appropriate, ensuring reliable transcription termination and sgRNA expression.
Through this cycle, we successfully established a CRISPR-Cas9-Am gene editing system suitable for Aureobasidium melanogenum, providing a robust tool for subsequent research.
Through two rounds of the DBTL cycle, we achieved the transition from the unsuccessful “ARS + gpdA” design to the successful “AMA1 + TEF1 + CYC1” iteration. This process not only optimized the experimental system for this study but also demonstrated the significant value of the DBTL engineering cycle in the development of synthetic biology tools for fungi.
To evaluate the practical applicability of the constructed CRISPR-Cas9-Am system in A. melanogenum, this study systematically compared the efficiencies of different gene knockout strategies.
Previously, the laboratory primarily relied on homologous recombination for gene editing. To evaluate the efficiency of the constructed CRISPR-Cas9-Am system, a comparative experiment was designed. A gene knockout vector based on homologous recombination was introduced into P16 to delete Ade2, and the knockout efficiency was assessed by PCR and gel electrophoresis using designed primers. The gel image is shown below.
The electrophoresis results showed that among 48 transformants, only 3 positive clones were detected, yielding a knockout efficiency of less than 10%, and the bands were faint. This indicates that the homologous recombination method has low efficiency and limited reproducibility in this host.
In the initially attempted CRISPR-Cas9 vector system based on the ARS autonomous replicating sequence and gpdA promoter, P16 transformants were screened on selective plates. None of the strains exhibited a red phenotype, indicating that no positive knockout transformants were obtained (0%). These results demonstrate that ARS and gpdA are poorly compatible in A. melanogenum and cannot achieve effective genome editing.
The gene knockout efficiencies of homologous recombination, the first-generation CRISPR-Cas9 knockout vector, and the CRISPR-Cas9-Am knockout vector are summarized below:
In summary, the comparison clearly shows that homologous recombination exhibits low efficiency, the first-generation vector is completely ineffective, while the improved CRISPR-Cas9-Am system demonstrates a significant advantage. This confirms that the system is highly efficient and practical in A. melanogenum, providing a powerful tool for subsequent gene function analysis and metabolic engineering applications.
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