Expression of CAS9 Nuclease of the CRISPR/Cas Genome Editing System in the Filamentous Fungus Penicillium verruculosum

The cas9 gene of the Streptococcus pyogenes bacterium, which encodes Cas9 nuclease of the class 2 CRISPR/Cas system, is expressed in the filamentous fungus Penicillium verruculosum under the control of the constitutive autologous promoter of the glyceraldehyde-3-phosphate dehydrogenase gpdA gene, wh...

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Bibliographic Details
Published in:Moscow University chemistry bulletin 2020-07, Vol.75 (4), p.243-249
Main Authors: Kislitsin, V. Yu, Chulkin, A. M., Sinelnikov, I. G., Sinitsyn, A. P., Rozhkova, A. M.
Format: Article
Language:English
Subjects:
Cellobiase
Chemistry
Chemistry and Materials Science
Chemistry/Food Science
CRISPR
Fluorescence
Fungi
Glucosidase
Nuclease
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Summary:The cas9 gene of the Streptococcus pyogenes bacterium, which encodes Cas9 nuclease of the class 2 CRISPR/Cas system, is expressed in the filamentous fungus Penicillium verruculosum under the control of the constitutive autologous promoter of the glyceraldehyde-3-phosphate dehydrogenase gpdA gene, which is cloned and sequenced for the first time in the present work. The functionality of the gpdA promoter is confirmed by the expression of the heterologous β-glucosidase gene of Aspergillus niger in P. verruculosum . The relative copy number and expression level of the cas9 gene in recombinant strains are determined by real-time PCR. In order to detect and determine the localization of Cas9 nuclease in P. verruculosum fungal cells, the chimeric Cas9 nuclease with the fluorescent eGFP protein added to the C-terminus was obtained. The fluorescence microscopy of the growing mycelium confirms the intracellular expression of the chimeric construct in the cells of recombinant strains.
ISSN:0027-1314
1935-0260
DOI:10.3103/S0027131420040033