Gene-editing Aspergillus Niger using CRISPR | High-efficiency & low off-target rate

Introduction:

Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called "black mold" on certain fruits and vegetables such as grapes, apricots, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called "black mold").

Aspergillus niger causes sooty mold of onions and ornamental plants. The infection of onion seedlings by A. niger can become systemic, manifesting only when conditions are conducive. A. niger causes a common postharvest disease of onions, in which the black conidia can be observed between the scales of the bulb. The fungus also causes disease in peanuts and grapes.

Aspergillus niger is less likely to cause human disease than some other Aspergillus species. In extremely rare instances, humans may become ill, but this is due to serious lung disease, aspergillosis, that can occur. Aspergillosis is, in particular, frequent among horticultural workers who inhale peat dust, which can be rich in Aspergillus spores. The fungus has also been found in the mummies of ancient Egyptian tombs and can be inhaled when they are disturbed. A. niger is one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss, and, only in severe cases, damage to the ear canal and tympanic membrane.

Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins;  other sources disagree, claiming this report is based upon a misidentification of the fungal species. Recent evidence suggests some true A. niger strains do produce ochratoxin A. It also produces the isoflavone orobol.

The filamentous fungus Aspergillus niger is an important cell factory used in the industry for the production of enzymes and organic acids. Owing to its genetic tractability, A. niger is widely used for research in fungal physiology, biochemistry, and biotechnology. The availability of the genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism. The utility of A. niger as an industrial cell factory and as a model organism for research can be further improved by the development of a high-efficiency genome-editing system. Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger services. However, the prices for gene knockout aspergillus niger are similar to those for cell lines.

 

Engineering Aspergillus niger for galactaric acid production

The use of CRISPR/Cas9 mediated gene deletion technology in A. niger in a metabolic engineering application. A transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and D-galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from D-galacturonic acid. The resulting strain was also converting pectin-rich biomass to galactaric acid in a consolidated bioprocess. As a result, a strain for the efficient production of galactaric acid from D-galacturonic acid was generated.

 

Engineering gRNA promoter using CRISPR/Cas9 in Aspergillus niger

In eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Researchers used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene-editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for the generation of designer chromosomes, as evidenced by the deletion of a 48 kb gene cluster required for the biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated the simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as a guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable the development of designer chromosomes in the model and industrially important fungi.

 

Performing CRISPR mutagenesis in Aspergillus using extra-chromosomal plasmid

With the ability of nucleases to make DNA double-strand break (DSB) in the host organism, nuclease-based gene targeting methods have been successfully used for gene disruption, knock-in mutation as well as improving heterologous protein production by integrating foreign genes to defined genomic loci. An efficient promoter that can facilitate gRNA transcription in vivo is a bottleneck of the adoption of the CRISPR/Cas9 system in Aspergilli. The method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Researchers reported a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. The results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.

 

Improving targeting efficiency in Aspergillus niger based on in vitro assembled ribonucleoproteins

In this study, a CRISPR/Cas9 facilitated transformation and genome editing method based on in vitro assembled ribonucleoprotein complexes was developed for the filamentous fungus Aspergillus niger. The method was downscaled to be compatible with 96-well microtiter plates. The optimized method resulted in 100% targeting efficiency for a single genomic target. After the optimization, the method was demonstrated to be suitable for multiplexed genome editing with two or three genomic targets in a metabolic engineering application. As a result, an A. niger price strain with improved capacity to produce galactarate, a potential chemical building block, was generated.

The developed microtiter plate compatible CRISPR/Cas9 method provides a basis for high-throughput genome editing workflows in A. niger and other related species. Besides, it improves the cost-effectiveness of CRISPR/Cas9 genome editing methods in fungi based on in vitro assembled ribonucleoproteins. The demonstrated metabolic engineering example with multiplexed genome editing highlights the applicability of the method.

 

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM) and knockin (KI).

 

References:

1.       Samson RA, Houbraken J, Summerbell RC, Flannigan B, Miller JD (2001). "Common and important species of fungi and actinomycetes in indoor environments". Microorganisms in Home and Indoor Work Environments. CRC. pp. 287–292. ISBN 978-0415268004.

2.       Kuivanen, J., Wang, Y.J. & Richard, P. Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9. Microb Cell Fact 15, 210 (2016).

3.       Xiaomei Zheng, Ping Zheng, Kun Zhang, Timothy C. Cairns, Vera Meyer, Jibin Sun, and Yanhe Ma. 5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger. ACS Synth. Biol. 2019, 8, 7, 1568–1574.

4.       Song L, Ouedraogo JP, Kolbusz M, Nguyen TTM, Tsang A. Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger. PLoS One. 2018;13(8):e0202868. Published 2018 Aug 24.

5.       Kuivanen, J., Korja, V., Holmström, S. et al. Development of microtiter plate scale CRISPR/Cas9 transformation method for Aspergillus niger based on in vitro assembled ribonucleoprotein complexes. Fungal Biol Biotechnol 6, 3 (2019).