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.

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 Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

Plants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 687 ◽  
Author(s):  
Chang Ho Ahn ◽  
Mummadireddy Ramya ◽  
Hye Ryun An ◽  
Pil Man Park ◽  
Yae-Jin Kim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genetically Modified Organisms ◽  
Ornamental Plants ◽  
Safety Evaluation ◽  
Evaluation Process ◽  
Vase Life ◽  
Transcription Activator ◽  
A Genome ◽  
Dna Specificity

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

TALEN-mediated genome editing: prospects and perspectives

2014 ◽  
Vol 462 (1) ◽  
pp. 15-24 ◽  
Author(s):  
David A. Wright ◽  
Ting Li ◽  
Bing Yang ◽  
Martin H. Spalding
Keyword(s):  
Genome Editing ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Natural Processes ◽  
Current State ◽  
A Genome ◽  
Dna Dsbs ◽  
Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

scholarly journals Highly efficient generation of bacteria leaf blight resistance and transgene-free rice using genome editing and multiplexed selection system

2020 ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Genomic Research ◽  
Blight Resistance ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Selection Of

Abstract Background Rice leaf blight is a worldwide devastating disease caused by bacteria Xanthomonas oryzae pv. Oryzae (Xoo). The UPT (up-regulated by transcription activator-like 1 effector) box in promoter region of the rice Xa13 gene played a key role in Xoo pathogenicity. Mutation of key bacterial protein binding site in UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistant to bacterial.Highly efficient generation and selection transgene-free, edited plants helpful to shorten and simple the gene editing breeding process. Selective elimination of transgenic pollen of E0 plants can enrich proportion of E1 transgene-free offspring and expression of the color mark gene in seeds makes the selection of E2 plants is very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacteria leaf blight resistance and transgene-free rice plants.Results We introduced site specific mutations into the UPT box using CRISPR/Cas12a technology to hamper TAL (Transcription-Activator Like effectors) protein binding and gene activation, and generated genome edited rice with improved bacteria blight resistance. Transgenic pollens of E0 plants were eliminated by pollen specific expression of α-amylase gene Zmaa1, the proportion of transgene-free plants were enriched from 25% to 50% in single T-DNA insertion events in E1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced 50% cost and achieved up to 98.64% of accuracy for selection of transgene-free edited plants. Conclusion We demonstrated core nucleotide deletion in the UPT box of Xa13 promoter conferred resistance to rice blight and selection of transgene-free plants were boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

scholarly journals Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wei Shen ◽  
Jun Zhang ◽  
Binan Geng ◽  
Mengyue Qiu ◽  
Mimi Hu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Strain Improvement ◽  
Genomic Research ◽  
Francisella Novicida ◽  
A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

scholarly journals Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae

2012 ◽  
Vol 78 (8) ◽  
pp. 2819-2829 ◽  
Author(s):  
Ryuta Wada ◽  
Jun-ichi Maruyama ◽  
Haruka Yamaguchi ◽  
Nanase Yamamoto ◽  
Yutaka Wagu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aspergillus Oryzae ◽  
Mating Type ◽  
Genetic Manipulation ◽  
Gene Replacement ◽  
Soy Sauce ◽  
Sexual Cycle ◽  
Dependent Manner ◽  
Content Type ◽  
Mating Type Genes

ABSTRACTThe potential for sexual reproduction inAspergillus oryzaewas assessed by investigating the presence and functionality ofMATgenes. Previous genome studies had identified aMAT1-1gene in the reference strain RIB40. We now report the existence of a complementaryMAT1-2gene and the sequencing of an idiomorphic region fromA. oryzaestrain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of theMAT1-1andMAT1-2genotypes among 180 strains assayed, including industrialtane-kojiisolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in whichMATgenes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both theMAT1-1andMAT1-2genes functionally regulate gene expression inA. oryzaein a mating type-dependent manner, the first such report for a supposedly asexual fungus.MAT1-1expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system inA. oryzae, and prospects for the discovery of a sexual cycle are discussed.

scholarly journals Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Transgenic Pollen ◽  
Selection Of

Abstract Background Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants. Results We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants. Conclusion We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

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