Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout
Bacillus licheniformis is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease,...
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KeAi Communications Co., Ltd.
2025-03-01
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| Series: | Synthetic and Systems Biotechnology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405805X24001133 |
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| author | Suxin Liu Fengxu Xiao Youran Li Yupeng Zhang Yanling Wang Guiyang Shi |
| author_facet | Suxin Liu Fengxu Xiao Youran Li Yupeng Zhang Yanling Wang Guiyang Shi |
| author_sort | Suxin Liu |
| collection | DOAJ |
| description | Bacillus licheniformis is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed Pmal for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in Bacillus licheniformis. Our system achieved a 100 % knockout efficiency for the single gene vpr and up to 80 % for simultaneous knockout of the double genes epr and mpr. Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by aprE contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by vpr, epr, and mpr genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms. |
| format | Article |
| id | doaj-art-be38478bb0434116b12c8c1c6c335de7 |
| institution | OA Journals |
| issn | 2405-805X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | KeAi Communications Co., Ltd. |
| record_format | Article |
| series | Synthetic and Systems Biotechnology |
| spelling | doaj-art-be38478bb0434116b12c8c1c6c335de72025-08-20T02:24:59ZengKeAi Communications Co., Ltd.Synthetic and Systems Biotechnology2405-805X2025-03-01101394810.1016/j.synbio.2024.08.002Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockoutSuxin Liu0Fengxu Xiao1Youran Li2Yupeng Zhang3Yanling Wang4Guiyang Shi5Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR China; Corresponding author. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR ChinaKey Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, PR China; Jiangsu Provincial Engineering Research Center for Bioactive Product Processing, Jiangnan University, Wuxi, 214122, Jiangsu, PR ChinaBacillus licheniformis is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance on resistance genes. Additionally, the established CRISPR gene editing technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple genes. To address this limitation, the CRISPR-Cpf1 system has been developed, enabling multiplexed gene editing across various microorganisms. Key to the efficient gene editing capability of this system is the rigorous screening of highly effective expression elements to achieve conditional expression of protein Cpf1. In this study, we employed mCherry as a reporter gene and harnessed Pmal for regulating the expression of Cpf1 to establish the CRISPR-Cpf1 gene editing system in Bacillus licheniformis. Our system achieved a 100 % knockout efficiency for the single gene vpr and up to 80 % for simultaneous knockout of the double genes epr and mpr. Furthermore, the culture of a series of protease-deficient strains revealed that the protease encoded by aprE contributed significantly to extracellular enzyme activity (approximately 80 %), whereas proteases encoded by vpr, epr, and mpr genes contributed to a smaller proportion of extracellular enzyme activity. These findings provide support for effective molecular modification and metabolic regulation in industrial organisms.http://www.sciencedirect.com/science/article/pii/S2405805X24001133Bacillus licheniformisGene editingCRISPR-Cpf1MCherry |
| spellingShingle | Suxin Liu Fengxu Xiao Youran Li Yupeng Zhang Yanling Wang Guiyang Shi Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout Synthetic and Systems Biotechnology Bacillus licheniformis Gene editing CRISPR-Cpf1 MCherry |
| title | Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout |
| title_full | Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout |
| title_fullStr | Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout |
| title_full_unstemmed | Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout |
| title_short | Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout |
| title_sort | establishment of the crispr cpf1 gene editing system in bacillus licheniformis and multiplexed gene knockout |
| topic | Bacillus licheniformis Gene editing CRISPR-Cpf1 MCherry |
| url | http://www.sciencedirect.com/science/article/pii/S2405805X24001133 |
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