One‐vector CRISPR/Cas9 genome engineering of the industrial fungus <i>Ashbya gossypii</i>

Jiménez, Alberto; Muñoz‐Fernández, Gloria; Ledesma‐Amaro, Rodrigo; Buey, Rubén M.; Revuelta, José Luis

doi:10.1111/1751-7915.13425

Cited by 21 publications

(25 citation statements)

References 31 publications

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“…When sgRNA2 (100 µg) targeting ura3 (Data S4) was delivered into pJW‐EXP‐intron‐opcas9, 28 transformants were obtained in 5‐FOA‐containing minimal medium (MM) plates. In Ashbya gossypii , different editing efficiency between the three ADE2 sgRNA‐dDNAs was also found (Jimenez et al , ). The observed difference in the disruption efficiency of ura3 may be due to the fact that the Cas9‐generated alleles display sequence‐dependent bias (Allen et al , ).…”

Section: Resultsmentioning

confidence: 93%

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Liu

Sun

You

et al. 2020

Microbial Biotechnology

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Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin‐resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9‐mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5’‐monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde‐3‐phosphate dehydrogenase gene intron in the Cas9 plasmid is 14–18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA‐directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.

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Section: Resultsmentioning

confidence: 93%

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Liu

Sun

You

et al. 2020

Microbial Biotechnology

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“…The CRISPR/Cpf1 system was assembled in a single vector containing all the required modules for genomic editions. The A. gossyppi CRISPR/Cas9 vector was used as a backbone that included the replication origins (yeast 2µ and bacterial ColE1) and the resistance markers (Amp R and G418 R ) [8]. The donor DNA and the modules for the expression of Cpf1 and crRNAs were assembled as follows: a synthetic codon-optimized ORF of the Cpf1 enzyme from Lachnospiraceae bacterium (LbCpf1) with a SV40 nuclear localization signal was assembled with the promoter and terminator sequences of the A. gossypii TSA1 and ENO1 genes, respectively.…”

Section: Assembly Of the Crispr/cpf1 System For A Gossypiimentioning

confidence: 99%

“…The CRISPR/Cas9 tool for A. gossypii, which was designed as a one-vector system, is optimized for the genomic engineering of multinucleated germlings of the fungus [8]. In order to expand the repertoire of genomic editing tools for A. gossypii, a CRISPR/Cpf1 system has been designed.…”

Section: Design Of a Crispr/cpf1 System Adapted For A Gossypiimentioning

confidence: 99%

“…In this context, the development of novel molecular tools is essential for the implementation of rational system metabolic engineering approaches in A. gossypii. Hence, a complete toolbox for genomic manipulation is available for A. gossypii, including a CRISPR/Cas9 system adapted for this fungus [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

“…The CRISPR/Cas9 system of A. gossypii shows a high editing efficiency for the introduction of gene deletions, insertions and nucleotide substitutions, which largely facilitates the genomic engineering of the fungus in a marker-less manner [8]. The efficiency of the system in a multinucleated syncytium such as the A. gossypii mycelia relies on a one-vector strategy that comprises the expression modules for the CAS9 and the synthetic guide RNA (sgRNA), and the donor DNA (dDNA).…”

Section: Introductionmentioning

confidence: 99%

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Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

Jiménez

Hoff

Revuelta

2019

Preprint

Self Cite

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The CRISPR/Cas technologies constitute an essential tool for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables the efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained to the existence of an specific 5'-NGG-3' PAM sequence in the target site.Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows to employ a T-rich PAM sequence (5'-TTTN-3') and facilitates the implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions into five different auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system was demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may significantly affect to the edition efficiency of the system.

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Unlocking Nature's Biosynthetic Power—Metabolic Engineering for the Fermentative Production of Chemicals

Hoff¹,

Plassmeier

Blankschien

et al. 2020

Angewandte Chemie

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Fermentation as a production method for chemicals is especially attractive, as it is based on cheap renewable raw materials and often exhibits advantages in terms of costs and sustainability. The tremendous development of technology in bioscience has resulted in an exponentially increasing knowledge about biological systems and has become the main driver for innovations in the field of metabolic engineering. Progress in recombinant DNA technology, genomics, and computational methods open new, cheaper, and faster ways to metabolically engineer microorganisms. Existing biosynthetic pathways for natural products, such as vitamins, organic acids, amino acids, or secondary metabolites, can be discovered and optimized efficiently, thereby enabling competitive commercial production processes. Novel biosynthetic routes can now be designed by the rearrangement of nature's unlimited number of enzymes and metabolic pathways in microbial strains. This expands the range of chemicals accessible by biotechnology and has yielded the first commercial products, while new fermentation technologies targeting novel active ingredients, commodity chemicals, and CO2‐fixation methods are on the horizon.

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One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

Cited by 21 publications

References 31 publications

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

Unlocking Nature's Biosynthetic Power—Metabolic Engineering for the Fermentative Production of Chemicals

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