Efficient gene deletion and replacement in Aspergillus niger by modified in vivo CRISPR/Cas9 systems
KeywordsAspergillus niger CRISPR/Cas9 Filamentous fungi Genome editing Ribozyme pAN7-1 Glucoamylase
clustered regularly interspaced short palindromic repeats
hepatitis delta virus
non-homologous DNA end joining
Aspergillus niger, as a member of filamentous fungi, is widely used to produce and secrete a variety of bioactive substance including organic acids, like citric acid (Wang et al. 2017), oxalic acid (Lee et al. 2018), gallic acid (Mata-Gomez et al. 2015) as well as many carbohydrate-active enzyme (CAzymes), such as glucoamylase (Suyama et al. 2017), glucose oxidase (Zhu et al. 2018), and amylase (Varalakshmi et al. 2009), which are valuable in industry especially food industry. And it is generally recognized as safe (GRAS) by the United States Food and Drug Administration. So, A. niger is considered to be one of the most important cell factories for industrial enzymes and organic acids production. Despite the importance of A. niger, the gene function and synthetic biology research have fallen behind because of the dominance of non-homologous DNA end joining (NHEJ) over homology directed repair (HDR) in filamentous fungi (Carvalho et al. 2010; Meyer et al. 2007).
In 1990s, deletion of Ku70/Ku80-protein complex was utilized to inactivate the NHEJ pathway, which was reported to strongly reduce the random integration of DNA fragments and lead to high homologous recombination efficiency in filamentous fungi. In A. niger, the deletion of kusA gene which encodes the ortholog of Ku70 protein strongly improved homologous recombination efficiency up to over 80% and did not influence the growth of A. niger (Weld et al. 2006). However, Δku70 was not easy to generate from wild type (WT) strain by traditional ways, and the mutants were further found to be more sensitive to some chemicals such as methyl mesylate, ethyl methane sulfonate and bleomycin (Meyer et al. 2007) so its application is still limited.
Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been discovered as an efficient gene editing technology in many organisms, such as plants (Liu et al. 2017; Shan et al. 2013), mice (Shen et al. 2013) and rice (Li et al. 2013). The nuclease Cas9 can cause a double-strand break (DSB) in the target deoxyribonucleic acid (DNA) which is formed by a 20-bp DNA sequence of target gene with a 5′-NGG nucleotide sequence in the subsequent downstream which is called the protospacer-adjacent motif (PAM) (Mei et al. 2016). Recently, the CRISPR/Cas9 system has been successfully applied in many filamentous fungi, such as Trichoderma reesei (Liu et al. 2015), Aspergillus oryzae (Katayama et al. 2016), Aspergillus fumigatus (Fuller et al. 2015), Neurospora crassa (Matsu-Ura et al. 2015), and Penicillium chrysogenum (Pohl et al. 2016). What is more, Nødvig et al. (2015) established a versatile CRISPR–Cas9 system with a special ribozyme–gRNA–ribozyme (RGR) structure for filamentous fungi which was benefit from the study in plant (Gao and Zhao 2014) and applied successfully in six different filamentous fungi consisting of A. niger. This structure effectively solved the problem of lacking promoters recognized by RNA polymerase III for the transcription of gRNA in A. niger. But the construction of the CRISPR–Cas9 vector depending on USER cloning or USER fusion (New England Biolabs, USA) raised the cost of the process. And the backbone plasmid pFC330 series in the paper is not common for most researchers. So, techniques based on common plasmid and easy-to-assemble elements still need to be further investigated.
In this study, we constructed a CRISPR/Cas9 tool plasmid based on general expression plasmid pAN7-1 for filamentous fungi and designed an RGR element in which the middle gRNA sequence can be easily changed by simple molecular cloning. The efficiency of using it to inactivate a pigment gene of olvA in A. niger was firstly investigated.
α-Glucosidase is a by-product of glucoamylase fermentation and its expression will reduce the activity and yield in the glucoamylase fermentation process of A. niger. So, disruption of α-glucosidase coding gene may benefit the production of glucoamylase. We chose one of the α-glucosidase family members agdF as the target gene for disruption because its expression level is the highest in the family during enzyme production process according to our previous transcriptome sequencing data (Lu et al. 2018). Replacement of agdF by glucoamylase gene using the CRISPR/Cas9 tool plasmid and a repair fragment was further tested.
Materials and methods
Strains and media
Escherichia coli strain DH5α was used to propagate all plasmids. The glucoamylase production A. niger strain CBS513.88 was preserved in our laboratory and used for genetic manipulation. PDA media (20% potato, 2% glucose, 2% agar) were used for sporulation of A. niger strains. CM medium (0.1% cosamino acids, 0.5% yeast extract, 2% 50% glucose mother liquor, 2% Asp + N, 0.2% 1 M MgSO4, 0.1% trace elements) was used for germination of spore. The transformants were grown on the following double layer medium. The upper media contained 0.95 M sucrose, 0.6% agar, 2% Asp + N, 0.2% 1 M MgSO4, 0.1% trace elements stock solution. The lower media contained 0.95 M sucrose, 1.2% agar, 2% Asp + N, 0.2% 1 M MgSO4, 0.1% trace elements. Asp + N (50 × stock solution): 3.5 M NaNO3, 0.35 M KCl, 0.55 M KH2PO4. Trace elements (1000 × stock solution): per liter, 10 g EDTA, 4.4 g ZnSO4·7H2O, 1.01 g MnCl2·4H2O, 0.32 g CoCl2·6H2O, 0.315 g CuSO4·5H2O, 0.22 g (NH4) 6Mo7O24·4H2O, 1.11 g CaCl2, 1 g FeSO4·7H2O).
Protoplast transformation of A. niger
Fungal spores were inoculated from glycerol stock on PDA culture plates and cultured at 30 °C for 4–5 days for the mycelium growth and spore generation. The fresh spores were eluted with 0.85% NaCl and 0.02% Tween 80. Spore suspension was inoculated into 250 ml liquid CM culture medium at a concentration of 2 × 108/ml for overnight cultivation (16–18 h) at 30 °C, 50 rpm. The germinated spores were harvested by filtering through three layers of miracloth (Millipore, cut and sterilized in advance), washed with SMC solution (1.33 M sorbitol, 50 mM CaCl2, 20 mM MES buffer, pH 5.8) and digested with enzyme solution (200 mg lysing enzyme with 10 ml SMC solution) for 3 h with rotatory shaking at speed of 75 rpm at 30 °C. Protoplast quality and quantity could be checked by microscopy during the digestion process. Before collecting protoplasts, 10 ml STC solution (1.33 M sorbitol, 50 mM CaCl2, 10 mM Tris/HCl, pH 7.5) was added to increase the release of protoplasts. Digested protoplasts were separated from cell wall debris by filtering through three layers of miracloth and the filtrate was precipitated at 3500 rpm for 10 min at 10 °C. The supernatant was discarded and the protoplasts were then washed 1-2 times with 1 ml STC solution at 4500 rpm for 5 min at 10 °C and resuspended in STC solution. The volume of STC solution was set according to the quantity of transformations (100 μl/transformation) and the final concentration of protoplast should be over 7 × 106/ml. Several 50 ml sterile centrifuge tubes were prepared, depending on the number of transformation experiments. For each transformation, 100 μl protoplast suspension, 8–10 μg of plasmid DNA and 25 μl PEG 6000 (25% polyethylene glycol 6000, dissolved at 65 °C in water bath for 10 min) were added to an empty 50 ml centrifuge tube and mixed softly. Subsequently, 1 ml PEG 6000 was added and evenly mixed into the mixture. Then, the tubes were incubated on ice for 5 min. 2 ml additional STC solution was added to the suspension and mixed slightly. Then, 10 ml upper medium containing hygromycin (100 μg/ml) was added into the suspension and mixed uniformly. The mixture was overlaid on the lower medium containing hygromycin (100 μg/ml), and the plates were put at 30 °C for 5–7 days allowing the growth of transformants.
Tool plasmid construction
The CRISPR/Cas9 expression vector pAN-olvA was constructed as follows. The Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system with a sequence encoding SV40 nuclear localization signal (SV40 NLS) at both 3′ and 5′-end was amplified from the plasmid pX458 (purchased from Addgene) by polymerase chain reaction (PCR) using primers Cas9-F and Cas9-R. The amplified Cas9 was inserted between the gpdA promoter (PgpdA) and trpC terminator (TtrpC) in pAN7-1 (purchased from Addgene) which was an expression plasmid widely used in filamentous fungi, yielding plasmid pANCas9. The pre-sgRNA fragment containing a hammerhead (HH) ribozyme at the 5′-end, the sequence-specific gRNA portion in the middle and a hepatitis delta virus (HDV) ribozyme at the 3′-end was obtained by synthesis (BGI, Shanghai). To facilitate the replacement of different sgRNA element targeting different genes in future, we added two unique endonuclease sites (HindIII and SpeI) at both ends of HH-sgRNA-HDV element by fusion PCR using primers (HindIII-sgRNA-F and sgRNA-SpeI-R). The CRISPR/Cas9 vector was assembled by One-Step Cloning Kit (Vazyme Biotech, China).
Over-expression of glaA gene and down-regulating agdF in one step by CRISPR
To reduce the expression of α-glucosidase and increase the expression of glucoamylase in the same time, we designed the one-step replacement strategy by CRISPR which utilized a plasmid with sgRNA targeting to agdF gene (pAN-agdF), a repair fragment containing over-expression cassette of glucoamylase encoding gene glaA and two 1 kb homologous arms flanking the agdF gene.
Primers used in the study
Efficient inactivation of pigment gene olvA in A. niger by CRISPR/Cas9 system embodied HH-sgRNA-HDV element
Mutated sequence of ΔolvA mutants
Over-expression of glaA and down-regulation of agdF in one step by CRISPR
Efficiency of gene replacement with different length of the homologous arm
In this study, the CRISPR/Cas9 system showed high efficiency for targeted gene inactivation and replacement in A. niger. Using this system, gene replacement can be introduced by homology arms as short as 100 bp. The results made the technique promising when compared to the reported efficiency of 80% in NHEJ-deficient A. niger using 500 bp homology arms (Meyer et al. 2007).
In this study, the Cas9 coding gene was from the Streptococcus pyogenes—without codon optimization, and the Cas9/sgRNA expression vector was derived from pAN7-1 which did not contain the AMA1 element necessary for extrachromosomal replication of a plasmid in filamentous fungi (Carvalho et al. 2010). It was undeniably that the copy numbers of Cas9 and sgRNA could be higher when carried by autonomously replicating plasmid. However, it has been considered that the amount of Cas9 protein and gRNA was not limiting factors for gene editing over many cases. Recently, Zheng et al. (2018) also reported their CRISPR/Cas9 gene editing system derived from pUC plasmid successfully worked in Aspergillus.
Although the RNA polymerase III promoters recently have been identified and used for expressing sgRNA in A. niger (Nødvig et al. 2018), RNA II promoters in RGR structure still have advantages. An important one is that some of them are inducible and can be regulated by environmental factors, such as carbon source and pH. This would benefit the expression of the RGR structure in special conditions and then control the function of the gene editing system in some extent.
The selection marker for plasmid construction and the length of repair fragment are among other issues that need to be considered when using this in vivo CRISPR system based on plasmid pAN7-1. In our study, there is no phenotype selection marker on the repair fragment, so the conidiophores germinated from primary positive colony on selective medium may have two types: correct mutants or off-targeted mutants in which the hygromycin resistant gene in plasmid integrated into another non-specific site. The two types can be further verified by PCR and DNA sequencing. For direct identification of gene knock-out, one antibiotic resistant gene for the Cas9/sgRNA expression plasmid and another antibiotic resistant gene or an auxotrophic marker gene for the repair fragment are recommended. However, a corresponding nutritional deficient host is needed for the latter condition.
In conclusion, we constructed a modified CRISPR/Cas9 system containing specific “RGR” structure and easy-to-switch element for a variety of target genes. The efficiency of the system was tested on the gene disruption of a pigment gene olvA in A. niger, and the mutation rate was up to 93%. In our gene replacement experiments targeting agdF genes for the enhanced expression of glaA gene, 3 secondary transformants out of 5 were confirmed to be the correct genotype and the activity of glucoamylase of agdF::glaA mutant was 25.9% higher than wild strain, while the activity of α-glucosidase of agdF::glaA mutant was 61.4% lower than wild strain. More interestingly, when the homologous arms of the repair fragment were shortened as 100 bp, the apparent recombination frequency is still high. The method may be applicable to more filamentous fungi that are difficult to genetically manipulate, such as Cephalosporium capitis (unpublished data), and so should have wide applications in genome editing of filamentous fungi.
YZ performed the experiments, analyzed data, and wrote the draft; LO designed the experiments and wrote the paper; YN and JC were involved in writing the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All materials are available in our lab.
Consent for publication
All the authors agree for publication.
Ethics approval and consent to participate
This work was financially supported by 2015 Open Project Fund of the State Key Laboratory of Bioreactor Engineering.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Lee SI, Lee KJ, Chun HH, Ha S, Gwak HJ, Kim HM, Lee JH, Choi HJ, Kim HH, Shin TS, Park HW, Kim JC (2018) Process development of oxalic acid production in submerged culture of Aspergillus niger F22 and its biocontrol efficacy against the root-knot nematode Meloidogyne incognita. Bioprocess Biosyst Eng 41:345–352CrossRefGoogle Scholar
- Lu H, Cao W, Liu X, Sui Y, Ouyang L, Xia J, Huang M, Zhuang Y, Zhang S, Noorman H, Chu J (2018) Multi-omics integrative analysis with genome-scale metabolic model simulation reveals global cellular adaptation of Aspergillus niger under industrial enzyme production condition. Sci Rep 8:14404CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.