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J Microbiol Methods ; 163: 105655, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31226337


Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.

Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050


Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.

Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
Springerplus ; 5(1): 1208, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27516946


OBJECTIVE: The aim of this study was to investigate the correlations between risk factors such as hypertension and the complex degrees of coronary arterial lesions (CAL). METHODS: We selected 462 patients with coronary heart disease (CHD) with confirmed the stenosis (≥50 %) in at least one major coronary artery on coronary angiography and divided them into the "CHD with hypertension" group (CHD-HT, n = 306) and the CHD group (n = 156). The characteristics of CAL and the occurrence of 2-year postoperative major adverse cardiac cerebrovascular events (MACCE) in the two groups were observed. RESULTS: The mean SYNTAX scores (SS) was higher in the CHD-HT group than in the CHD group (P < 0.05). The proportions of complex, calcified, and diffused long lesions in the PCI patients' target vascular lesions, as well as the total MACCE incidence, were significantly higher in the CHD-HT group than in the CHD group (P < 0.05). Logistic multifactor regression analysis showed that age, male sex, hypertension, diabetes, hyperlipidemia, and previous history of myocardial infarction were positively correlated with the SS (P < 0.05). CONCLUSIONS: The patients with CHD-HT exhibited complicated and diffused CAL, and arterial hypertension can be considered as a risk factor for the complexity of coronary lesions in patients with ischemic heart disease.