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CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae

Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene func...

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Autores principales: Li, Qinghua, Lu, Jinchang, Zhang, Guoqiang, Zhou, Jingwen, Li, Jianghua, Du, Guocheng, Chen, Jian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9864741/
https://www.ncbi.nlm.nih.gov/pubmed/36675930
http://dx.doi.org/10.3390/jof9010109
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author Li, Qinghua
Lu, Jinchang
Zhang, Guoqiang
Zhou, Jingwen
Li, Jianghua
Du, Guocheng
Chen, Jian
author_facet Li, Qinghua
Lu, Jinchang
Zhang, Guoqiang
Zhou, Jingwen
Li, Jianghua
Du, Guocheng
Chen, Jian
author_sort Li, Qinghua
collection PubMed
description Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins.
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spelling pubmed-98647412023-01-22 CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae Li, Qinghua Lu, Jinchang Zhang, Guoqiang Zhou, Jingwen Li, Jianghua Du, Guocheng Chen, Jian J Fungi (Basel) Article Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins. MDPI 2023-01-13 /pmc/articles/PMC9864741/ /pubmed/36675930 http://dx.doi.org/10.3390/jof9010109 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Li, Qinghua
Lu, Jinchang
Zhang, Guoqiang
Zhou, Jingwen
Li, Jianghua
Du, Guocheng
Chen, Jian
CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title_full CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title_fullStr CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title_full_unstemmed CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title_short CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
title_sort crispr/cas9-mediated multiplexed genome editing in aspergillus oryzae
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9864741/
https://www.ncbi.nlm.nih.gov/pubmed/36675930
http://dx.doi.org/10.3390/jof9010109
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