Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system
Ting Zeng,
Jie Yin,
Ziwen Liu,
Zhaoxing Li,
Yu Zhang,
Yang Lv,
Mei-Ling Lu,
Min Luo,
Meirong Chen,
Yibei Xiao
Affiliations
Ting Zeng
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Jie Yin
Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Ziwen Liu
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Zhaoxing Li
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Yu Zhang
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Yang Lv
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
Mei-Ling Lu
Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Min Luo
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
Meirong Chen
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China; Corresponding author
Yibei Xiao
State 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 author
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.