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V. Yu. Kislitsin, A. M. Chulkin, I. G. Sinelnikov, A. P. Sinitsyn, A. M. Rozhkova
Expression of Cas9
nuclease of CRISPR/Cas genome editing system in filamentous fungi penicillium
verruculosum
Abstract
The cas9 gene of the Streptococcus
pyogenes bacterium, which encodes second-class CRISPR / Cas system Cas9
nuclease, was expressed in filamentous fungus Penicillium verruculosum
under the control of the constitutive autologous promoter of the gpdA
gene encoding glyceraldehyde 3-phosphate dehydrogenase, which was cloned and
sequenced at the first time in the this work. The functionality of the gpdA
promoter was confirmed by expression of the heterologous Aspergillus nigerβ-glucosidase in P. verruculosum. The
relative copy number and expression level of the cas9 gene in
recombinant strains was counted by real-time PCR. To detect the localization
of Cas9 nuclease in P. verruculosum fungal cells a fluorescent eGFP protein
was added to the C-terminus of Cas9 and the chimeric nuclease was expressed.
Fluorescence microscopy of growing mycelium confirmed the intracellular
expression of the chimeric construct in cells of
recombinant strains.
Key words:
genome editing, Penicillium verruculosum, gpdA promoter.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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