Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system
Summary: The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associ...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-07-01
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| Series: | Cell Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S221112472300709X |
| _version_ | 1797794006311108608 |
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| author | Ting Zeng Jie Yin Ziwen Liu Zhaoxing Li Yu Zhang Yang Lv Mei-Ling Lu Min Luo Meirong Chen Yibei Xiao |
| author_facet | Ting Zeng Jie Yin Ziwen Liu Zhaoxing Li Yu Zhang Yang Lv Mei-Ling Lu Min Luo Meirong Chen Yibei Xiao |
| author_sort | Ting Zeng |
| collection | DOAJ |
| description | Summary: The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associates with AAA+ ATPase TnsC and catalyzes donor DNA cleavage and integration remains ambiguous. In this study, we demonstrate that TniQ-dCas9 fusion can direct site-specific transposition by TnsB/TnsC in ShCAST. TnsB is a 3′-5′ exonuclease that specifically cleaves donor DNA at the end of the terminal repeats and integrates the left end prior to the right end. The nucleotide preference and the cleavage site of TnsB are markedly different from those of the well-documented MuA. We also find that TnsB/TnsC association is enhanced in a half-integration state. Overall, our results provide valuable insights into the mechanism and application expansion of CRISPR-mediated site-specific transposition by TnsB/TnsC. |
| first_indexed | 2024-03-13T02:56:32Z |
| format | Article |
| id | doaj.art-7703c8b65b2b43d38a6ca514fcc36006 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-03-13T02:56:32Z |
| publishDate | 2023-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-7703c8b65b2b43d38a6ca514fcc360062023-06-28T04:29:28ZengElsevierCell Reports2211-12472023-07-01427112698Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST systemTing Zeng0Jie Yin1Ziwen Liu2Zhaoxing Li3Yu Zhang4Yang Lv5Mei-Ling Lu6Min Luo7Meirong Chen8Yibei Xiao9State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, ChinaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, ChinaDepartment of Biological Sciences, National University of Singapore, Singapore 117543, SingaporeState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China; Corresponding authorState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China; Chongqing Innovation Institute of China Pharmaceutical University, Chongqing 401135, China; Corresponding authorSummary: The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associates with AAA+ ATPase TnsC and catalyzes donor DNA cleavage and integration remains ambiguous. In this study, we demonstrate that TniQ-dCas9 fusion can direct site-specific transposition by TnsB/TnsC in ShCAST. TnsB is a 3′-5′ exonuclease that specifically cleaves donor DNA at the end of the terminal repeats and integrates the left end prior to the right end. The nucleotide preference and the cleavage site of TnsB are markedly different from those of the well-documented MuA. We also find that TnsB/TnsC association is enhanced in a half-integration state. Overall, our results provide valuable insights into the mechanism and application expansion of CRISPR-mediated site-specific transposition by TnsB/TnsC.http://www.sciencedirect.com/science/article/pii/S221112472300709XCP: Molecular biologyCP: Microbiology |
| spellingShingle | Ting Zeng Jie Yin Ziwen Liu Zhaoxing Li Yu Zhang Yang Lv Mei-Ling Lu Min Luo Meirong Chen Yibei Xiao Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system Cell Reports CP: Molecular biology CP: Microbiology |
| title | Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system |
| title_full | Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system |
| title_fullStr | Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system |
| title_full_unstemmed | Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system |
| title_short | Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system |
| title_sort | mechanistic insights into transposon cleavage and integration by tnsb of shcast system |
| topic | CP: Molecular biology CP: Microbiology |
| url | http://www.sciencedirect.com/science/article/pii/S221112472300709X |
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