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Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes.
J Microbiol Methods. 2019 08; 163:105655.JM

Abstract

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.

Authors+Show Affiliations

School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology Guangzhou, Guangzhou 510006, Guangdong, China.School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology Guangzhou, Guangzhou 510006, Guangdong, China.School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology Guangzhou, Guangzhou 510006, Guangdong, China.School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology Guangzhou, Guangzhou 510006, Guangdong, China. Electronic address: btbinwang@scut.edu.cn.School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China; Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology Guangzhou, Guangzhou 510006, Guangdong, China. Electronic address: btlipan@scut.edu.cn.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31226337

Citation

Dong, Hongzhi, et al. "Efficient Genome Editing in Aspergillus Niger With an Improved Recyclable CRISPR-HDR Toolbox and Its Application in Introducing Multiple Copies of Heterologous Genes." Journal of Microbiological Methods, vol. 163, 2019, p. 105655.
Dong H, Zheng J, Yu D, et al. Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes. J Microbiol Methods. 2019;163:105655.
Dong, H., Zheng, J., Yu, D., Wang, B., & Pan, L. (2019). Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes. Journal of Microbiological Methods, 163, 105655. https://doi.org/10.1016/j.mimet.2019.105655
Dong H, et al. Efficient Genome Editing in Aspergillus Niger With an Improved Recyclable CRISPR-HDR Toolbox and Its Application in Introducing Multiple Copies of Heterologous Genes. J Microbiol Methods. 2019;163:105655. PubMed PMID: 31226337.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes. AU - Dong,Hongzhi, AU - Zheng,Junwei, AU - Yu,Dou, AU - Wang,Bin, AU - Pan,Li, Y1 - 2019/06/18/ PY - 2019/01/31/received PY - 2019/06/13/revised PY - 2019/06/13/accepted PY - 2019/6/22/pubmed PY - 2020/6/17/medline PY - 2019/6/22/entrez KW - Aspergillus niger KW - CRISPR-HDR tool box KW - Donor DNA KW - Exocellular protein expression KW - Gene knock-in KW - Multiple-site gene edit SP - 105655 EP - 105655 JF - Journal of microbiological methods JO - J. Microbiol. Methods VL - 163 N2 - 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. SN - 1872-8359 UR - https://www.unboundmedicine.com/medline/citation/31226337/Efficient_genome_editing_in_Aspergillus_niger_with_an_improved_recyclable_CRISPR_HDR_toolbox_and_its_application_in_introducing_multiple_copies_of_heterologous_genes_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0167-7012(19)30096-X DB - PRIME DP - Unbound Medicine ER -