Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae

2015 ◽  
Vol 38 (4) ◽  
pp. 637-642 ◽  
Author(s):  
Takuya Katayama ◽  
Yuki Tanaka ◽  
Tomoya Okabe ◽  
Hidetoshi Nakamura ◽  
Wataru Fujii ◽  
...  
Keyword(s):  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
A Genome

scholarly journals Genome Editing Technology and Its Application Potentials in the Industrial Filamentous Fungus Aspergillus oryzae

2021 ◽  
Vol 7 (8) ◽  
pp. 638
Author(s):  
Jun-ichi Maruyama
Keyword(s):  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Genetic Manipulation ◽  
Wild Strain ◽  
Soy Sauce ◽  
Transcription Activator ◽  
Gene Modification ◽  
A Genome ◽  
Industrial Strains

Aspergillus oryzae is a filamentous fungus that has been used in traditional Japanese brewing industries, such as the sake, soy sauce, and miso production. In addition, A. oryzae has been used in heterologous protein production, and the fungus has been recently used in biosynthetic research due to its ability to produce a large amount of heterologous natural products by introducing foreign biosynthetic genes. Genetic manipulation, which is important in the functional development of A. oryzae, has mostly been limited to the wild strain RIB40, a genome reference suitable for laboratory analysis. However, there are numerous industrial brewing strains of A. oryzae with various specialized characteristics, and they are used selectively according to the properties required for various purposes such as sake, soy sauce, and miso production. Since the early 2000s, genome editing technologies have been developed; among these technologies, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have been applied to gene modification in A. oryzae. Notably, the CRISPR/Cas9 system has dramatically improved the efficiency of gene modification in industrial strains of A. oryzae. In this review, the development of genome editing technology and its application potentials in A. oryzae are summarized.

scholarly journals Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Takuya Katayama ◽  
Hidetoshi Nakamura ◽  
Yue Zhang ◽  
Arnaud Pascal ◽  
Wataru Fujii ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Gene Deletion ◽  
Multiple Gene ◽  
Content Type ◽  
Industrial Strains ◽  
Marker Free

ABSTRACT Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

scholarly journals Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

2021 ◽  
Vol 22 (2) ◽  
pp. 480
Author(s):  
Jongrae Kim ◽  
Kwang Suk Chang ◽  
Sangmuk Lee ◽  
EonSeon Jin
Keyword(s):  
Genome Editing ◽  
Chlorella Vulgaris ◽  
Negative Selection ◽  
Screening Strategy ◽  
Feed Additive ◽  
Cell Factory ◽  
Dna Transformation ◽  
Research Groups ◽  
A Genome ◽  
Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

A genome-editing off switch

2016 ◽  
Vol 18 (2) ◽  
pp. 69-69
Author(s):  
Ross Cloney
Keyword(s):  
Genome Editing ◽  
A Genome

Nanopore sequencing reveals a structural alteration of mirror‐image duplicated genes in a genome‐editing mouse line

2019 ◽  
Vol 60 (4) ◽  
pp. 120-125 ◽  
Author(s):  
Sachiko Miyamoto ◽  
Kazushi Aoto ◽  
Takuya Hiraide ◽  
Mitsuko Nakashima ◽  
Shuji Takabayashi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mirror Image ◽  
Structural Alteration ◽  
Nanopore Sequencing ◽  
Duplicated Genes ◽  
Mouse Line ◽  
A Genome

CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

2016 ◽  
Vol 45 (24) ◽  
pp. 6666-6684 ◽  
Author(s):  
Amrita Singh ◽  
Debojyoti Chakraborty ◽  
Souvik Maiti
Keyword(s):  
Genome Editing ◽  
Chemical Biology ◽  
Genome Engineering ◽  
A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

scholarly journals A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy

2020 ◽  
Author(s):  
Jichao Wang ◽  
Xinyue Sui ◽  
Yamei Ding ◽  
Yingxin Fu ◽  
Xinjun Feng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Genome Editing ◽  
The Other ◽  
Plasmid Curing ◽  
Gene Deletions ◽  
A Genome ◽  
Single Clone ◽  
Previous Round ◽  
Genome Modifications

Abstract The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

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