Editing Aspergillus terreus using the CRISPR-Cas9 system.

Autor: Shih SY; Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung City, Taiwan., Mortensen UH; DTU Bioengineering, Technical University of Denmark, Lyngby, Denmark., Chang FR; Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung City, Taiwan.; Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City, Taiwan., Tsai H; Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung City, Taiwan.; Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung City, Taiwan.
Jazyk: angličtina
Zdroj: Synthetic biology (Oxford, England) [Synth Biol (Oxf)] 2022 Dec 06; Vol. 7 (1), pp. ysac031. Date of Electronic Publication: 2022 Dec 06 (Print Publication: 2022).
DOI: 10.1093/synbio/ysac031
Abstrakt: CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system to various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in Aspergillus terreus . In this study, we present CRISPR/Cas9 genome editing in A. terreus . To optimize the guide ribonucleic acid (gRNA) expression, we constructed a modified single-guide ribonucleic acid (sgRNA)/Cas9 expression plasmid. By co-transforming an sgRNA/Cas9 expression plasmid along with maker-free donor deoxyribonucleic acid (DNA), we precisely disrupted the lovB and lovR genes, respectively, and created targeted gene insertion ( lovF gene) and iterative gene editing in A. terreus ( lovF and lovR genes). Furthermore, co-delivering two sgRNA/Cas9 expression plasmids resulted in precise gene deletion (with donor DNA) in the ku70 and pyrG genes, respectively, and efficient removal of the DNA between the two gRNA targeting sites (no donor DNA) in the pyrG gene. Our results showed that the CRISPR/Cas9 system is a powerful tool for precise genome editing in A. terreus , and our approach provides a great potential for manipulating targeted genes and contributions to gene functional study of A. terreus .
(© The Author(s) 2022. Published by Oxford University Press.)
Databáze: MEDLINE
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