Expression of CAS9 Nuclease of the CRISPR/Cas Genome Editing System in the Filamentous Fungus Penicillium verruculosum
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ession of CAS9 Nuclease of the CRISPR/Cas Genome Editing System in the Filamentous Fungus Penicillium verruculosum V. Yu. Kislitsina, A. M. Chulkina, I. G. Sinelnikova, A. P. Sinitsyna, b, and A. M. Rozhkovaa, * aFederal
Research Center “Fundamentals of Biotechnology,” Russian Academy of Sciences, Moscow, Russia b Department of Chemistry, Moscow State University, Moscow, Russia *e-mail: [email protected] Received January 10, 2020; revised January 12, 2020; accepted January 20, 2020
Abstract—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. Keywords: genome editing, Penicillium verruculosum, gpdA promoter DOI: 10.3103/S0027131420040033
INTRODUCTION Genome editing methods are being increasingly used in biotechnology. This approach allows knocking out certain genes, removing entire regions of the genome or substituting its regions with donor DNA fragments [1, 2]. The method of genome editing is based on the use of intracellular mechanisms of repairing and recombining genomic DNA. It is possible to effectively activate these processes in the desired regions of the genome by generating double-stranded breaks of genomic DNA in these sites using artificial programmable nucleases [3]. One of the most used nucleases of this type is the Cas9 protein in the complex with single guide RNA (sgRNA). The widespread use of this complex is determined by the convenience of working with it rather than programmable nucleases of other types [4]. The specificity of the site of DNA hydrolysis by the Cas9-sgRNA complex is determined by the sequence of the 20-nucleotide region of the guide RNA, called the protospacer, which is complementary to the edited fragment of the genome. Thus, to obtain a specific double-stranded break with Cas9 nuclease, it is only required to deliver this protein to the cell with the guide RNA of approximately 100 nucleotides in length or to obtain them in vivo. The filamentous fungus genome was first edited using the Cas9 nuclease in 2015 [5]. The application of this approach greatly facilitates the study of gene func-
tions, as well as the production of industrial strains with improved tech
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