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.

中文翻译：

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CRISPR / Cas基因组编辑系统CAS9核酸酶在丝状真菌青霉菌中的表达

摘要

化脓性链球菌细菌的cas9基因编码2类CRISPR / Cas系统的Cas9核酸酶，在丝状真菌Penicillium v​​erruculosum中表达，该酶由甘油醛-3-磷酸脱氢酶gpdA基因的组成型自体启动子控制。在本工作中是第一次克隆和测序。所述的功能性gpdA启动子由异源β葡糖苷酶基因的表达证实黑曲霉在P. verruculosum。cas9的相对拷贝数和表达水平通过实时PCR确定重组菌株中的基因。为了检测和确定疣状假单胞菌真菌细胞中Cas9核酸酶的定位，获得了带有荧光eGFP蛋白的C9嵌合Cas9核酸酶。生长的菌丝体的荧光显微镜检查证实了嵌合构建体在重组菌株细胞中的细胞内表达。