A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster
The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct ca...
| Published in: | Microorganisms |
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| Main Authors: | , , , , , , |
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-07-01
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| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/12/7/1462 |
| _version_ | 1850133022319312896 |
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| author | Yujing Guo Guang Cai Huiying Li Zhenquan Lin Shuobo Shi Jin Jin Zihe Liu |
| author_facet | Yujing Guo Guang Cai Huiying Li Zhenquan Lin Shuobo Shi Jin Jin Zihe Liu |
| author_sort | Yujing Guo |
| collection | DOAJ |
| container_title | Microorganisms |
| description | The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from <i>Streptomyces ceruleus</i> A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from <i>B. subtilis</i> 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources. |
| format | Article |
| id | doaj-art-163145bcd8fc4ece8de09a8f9c10aeb9 |
| institution | Directory of Open Access Journals |
| issn | 2076-2607 |
| language | English |
| publishDate | 2024-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| spelling | doaj-art-163145bcd8fc4ece8de09a8f9c10aeb92025-08-19T23:52:06ZengMDPI AGMicroorganisms2076-26072024-07-01127146210.3390/microorganisms12071462A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene ClusterYujing Guo0Guang Cai1Huiying Li2Zhenquan Lin3Shuobo Shi4Jin Jin5Zihe Liu6State Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaState Key Laboratory of Chemical Resource Engineering, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, ChinaThe ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from <i>Streptomyces ceruleus</i> A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from <i>B. subtilis</i> 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.https://www.mdpi.com/2076-2607/12/7/1462CRISPR/Cas9<i>in vitro</i> DNA assemblysynthetic genomesbiosynthetic gene cluster |
| spellingShingle | Yujing Guo Guang Cai Huiying Li Zhenquan Lin Shuobo Shi Jin Jin Zihe Liu A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster CRISPR/Cas9 <i>in vitro</i> DNA assembly synthetic genomes biosynthetic gene cluster |
| title | A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster |
| title_full | A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster |
| title_fullStr | A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster |
| title_full_unstemmed | A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster |
| title_short | A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster |
| title_sort | crispr cas9 mediated large fragment assembly method for cloning genomes and biosynthetic gene cluster |
| topic | CRISPR/Cas9 <i>in vitro</i> DNA assembly synthetic genomes biosynthetic gene cluster |
| url | https://www.mdpi.com/2076-2607/12/7/1462 |
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