Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope

Xu Feng (), Ruyi Xu, Jianglan Liao, Jingyu Zhao, Baochang Zhang, Xiaoxiao Xu, Pengpeng Zhao, Xiaoning Wang, Jianyun Yao, Pengxia Wang, Xiaoxue Wang, Wenyuan Han and Qunxin She ()
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Xu Feng: Shandong University
Ruyi Xu: Shandong University
Jianglan Liao: Shandong University
Jingyu Zhao: Shandong University
Baochang Zhang: Shandong University
Xiaoxiao Xu: Shandong University
Pengpeng Zhao: Shandong University
Xiaoning Wang: Shandong University
Jianyun Yao: Chinese Academy of Sciences
Pengxia Wang: Chinese Academy of Sciences
Xiaoxue Wang: Chinese Academy of Sciences
Wenyuan Han: Huazhong Agricultural University
Qunxin She: Shandong University

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

Date: 2024
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DOI: 10.1038/s41467-024-47697-4

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