CRISPR/Cas9‐based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi

Ullah, Mati; Lin, Xia; Xie, Shangxian; Sun, Su

doi:10.1002/bab.2077

Cited by 25 publications

(20 citation statements)

References 161 publications

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“…Once the DNA double-strand is cleaved at the target site by Cas9 nuclease activity, both ends can subsequently be repaired by the non-homologous end joining (NHEJ) or homology directed repair (HDR) pathway. Due to the simplicity, efficiency, and the possibility of introducing mutations in multiple genes in one step, CRISPR/Cas9 has become a fast-growing genome editing tool in fungi [19]. It has been developed into a powerful technology that has been applied to various filamentous fungi, including A. nidulans and A. aculeatus [20], A. oryzae [21], A. fumigatus [22,23], A. niger [24], A. carbonarius [25], Neurospora crassa [26], Alternaria alternata [27], Fusarium oxysporum [28], and F. graminearum [29].…”

Section: Introductionmentioning

confidence: 99%

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Ferrara

Gallo

Cervini

et al. 2021

Toxins

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Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

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Section: Introductionmentioning

confidence: 99%

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Ferrara

Gallo

Cervini

et al. 2021

Toxins

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“…(ii) Generally, the sgRNA can be expressed both in vitro and in vivo, but the in vitro transcription and purification process is laborious and costly. (iii) RNA polymerase type III promoters (U6 and U3 promoters) are frequently applied for the in vivo transcription of the sgRNA [ 41 ]. Unfortunately, these kinds of promoters are ill-defined in filamentous fungi [ 42 ].…”

Section: Discussionmentioning

confidence: 99%

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

Chen

Cai

Yang

et al. 2022

JoF

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Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding cas9, a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene rpd3 was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.

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“…Besides selection markers, and transformation strategies discussed before, genetic manipulations is a successful strategy used to investigate gene functions or enhance the ability of a strain to produce a certain product (Son and Park 2020;Ullah et al 2020). To do so, it is important to generate a host with effective selection markers and high transformation rate.…”

Section: Genetic Manipulationsmentioning

confidence: 99%

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool

Daba

Mostafa

Elkhateeb

2021

Bioresour. Bioprocess.

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Aspergillus oryzae (A. oryzae) is a filamentous micro-fungus that is used from centuries in fermentation of different foods in many countries all over the world. This valuable fungus is also a rich source of many bioactive secondary metabolites. Moreover, A. oryzae has a prestigious secretory system that allows it to secrete high concentrations of proteins into its culturing medium, which support its use as biotechnological tool in veterinary, food, pharmaceutical, and industrial fields. This review aims to highlight the significance of this valuable fungus in food industry, showing its generosity in production of nutritional and bioactive metabolites that enrich food fermented by it. Also, using A. oryzae as a biotechnological tool in the field of enzymes production was described. Furthermore, domestication, functional genomics, and contributions of A. oryzae in functional production of human pharmaceutical proteins were presented. Finally, future prospects in order to get more benefits from A. oryzae were discussed.

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CRISPR/Cas9‐based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi

Cited by 25 publications

References 161 publications

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

The ancient koji mold (Aspergillus oryzae) as a modern biotechnological tool

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