Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA

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Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA

Data availability

The structures of Cas12p–TrxA–sgRNA–target DNA have been deposited in the Protein Data Bank under the accession numbers 9JFS and 9JG3, and in the EMDB under the accession numbers EMD-61438 and EMD-61449. The next-generation sequencing data containing PAM-depletion and RNA sequencing raw reads are available on the Sequence Read Archive (SRA) under BioProject PRJNA1159697 and PRJNA1159999. The raw data of MS and PPI screening are provided in Supplementary Data 2 and 3, respectively. Source data are provided with this paper.

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Acknowledgements

This work was financially supported by grant nos. 22525702 and 22277078 from the National Natural Science Foundation of China, grant nos. 2022YFC3403400 and 2023YFC3400200 from the National Key R&D Program of China, grant no. 23HC1400800 from the Shanghai Committee of Science and Technology, China, grant nos. 2023M742367 and 2024M752064 from China Postdoctoral Science Foundation, grant no. GZC20231672 from Postdoctoral Fellowship Program of CPSF, grant no. SPST-YSFZ-2024-01 from School of Physical Science and Technology, ShanghaiTech University and grant no. 2025AI4S04002 from ShanghaiTech AI4S Initiative, ShanghaiTech University. We thank the Bio-EM facility at ShanghaiTech University for cryo-EM data collections, and we are grateful to L. Wang and D. Liu for their help with cryo-EM technical support. In addition, we also thank the staff members of the Electron Microscopy System at the NFPS in Shanghai for providing technical assistance in data collection. We thank C. Su of the MS System at the NFPS, Shanghai Advanced Research Institute, Chinese Academy of Science, China, for mass spectrometry sample preparation, data collection and data analysis. The work was supported by HPC Platform of ShanghaiTech University. ChatGPT-4 was used for language editing.

Author information

Author notes

  1. These authors contributed equally: Zhipeng Wang, Yujue Wang.

Authors and Affiliations

  1. School of Physical Science and Technology and State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai, China

    Zhipeng Wang, Yujue Wang, Hui Gao, Jiani Dai, Na Tang, Yannan Wang & Quanjiang Ji

  2. Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Quanjiang Ji

  3. Shanghai Clinical Research and Trial Center, Shanghai, China

    Quanjiang Ji

Authors

  1. Zhipeng Wang
  2. Yujue Wang
  3. Hui Gao
  4. Jiani Dai
  5. Na Tang
  6. Yannan Wang
  7. Quanjiang Ji

Contributions

Q.J. directed the project. Q.J., Z.W. and Yujue Wang conceived the project. Z.W. established the CRISPR–Cas12 systems mining pipeline and conducted the PPIs and bioinformatics analyses. Z.W. and Yujue Wang constructed the plasmids and performed the PAM-depletion and small RNA-sequencing assays. Yujue Wang performed the phage plaque, pull-down, in vitro FQ, T4Cas12p recombinant phage construction and in vitro DNA cleavage assays. Yujue Wang, Z.W., H.G. and J.D. performed the genome interference assay. Yujue Wang, Z.W., H.G. and Yannan Wang performed the protein purification. Z.W., Yujue Wang, N.T., H.G. and J.D. prepared the cryo-EM samples and Z.W. determined the structures. Q.J., Z.W. and Yujue Wang analysed and discussed the experimental data. Z.W. and Yujue Wang prepared the figures. Q.J., Z.W. and Yujue Wang wrote the paper.

Corresponding author

Correspondence to Quanjiang Ji.

Ethics declarations

Competing interests

Q.J., Z.W. and Yujue Wang have filed a patent application related to this work through ShanghaiTech University (2024119245216). Q.J. is the scientific founder of Castalysis Bioscience. The other authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Chase Beisel, Alex Johnson, John van der Oost and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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Extended data

Extended Data Fig. 1 PAM preferences and biochemical properties of Cas12p.

a, 4 N Heatmap of PAM sequence for Cas12p-1 (left) and Cas12p-2 (right). Cas12p-1 shows a higher PAM depletion efficiency than Cas12p-2. b, In vitro dsDNA cleavage with four different PAMs by Cas12p-1. The order of the preferred PAM sequence is GTA > GTG > GTT > GTC. c, d, Schematics and results of small RNA sequencing for the CRISPR-Cas12p from E. coli heterologous expression (c) or RNP pulldown (d). e, Detailed cleavage kinetics of Cas12p under various conditions. This panel presents the cleavage efficiency of Cas12p across different experimental variables, including temperature, the presence of divalent metal ions, concentrations of Mg2+ and Mn2+, and NaCl concentration, offering insights into the optimal biochemical conditions for its activity. Data are shown as mean ± s.d. of three biological replicates. f,g, TBE-Urea-PAGE assays demonstrate the cleavage patterns of FAM/Texas Red-labelled TS DNA (f) and NTS DNA (g) by Cas12p. h, Trans-cleavage activity evaluation of Cas12p using in vitro fluorescence-quenching assay. RFU, Relative Fluorescence Units. Data are shown as mean ± s.d. of three biological replicates.

Source data

Extended Data Fig. 2 Cryo-EM data analysis of the Cas12p-TrxA-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex.

a, SDS-PAGE (stained with CBB, left) and Urea-PAGE (right) analysis of the Cas12p-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex. b, Size-exclusion chromatography (SEC) profile of Cas12p-sgRNA RNP (left) and the Cas12p-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex (right). c, Cryo-EM data processing workflow for the Cas12p-TrxA-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex. d, Representative cryo-EM micrograph from a total of 3976 micrographs. e, Representative 2D class averages. f, The half-map Fourier shell correlation (FSC) curves. g, Direction distribution plot. h, Cryo-EM density map coloured by local resolution.

Source data

Extended Data Fig. 3 Complex structures of different proteins and thioredoxin.

a, Close-up view of the predicted loop structure (goldenrod) and the unexpected globular density (violet mesh) of Cas12p-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex. b, Pull-down assay using untagged Cas12p alone as the negative control. c, Top-ranking model of Cas12p-TrxA complex predicted by AlphaFold-Multimer. The structural model was coloured by pLDDT scores. TrxA is displayed within an orchid transparent surface. d–g, Structures of the PAPS reductase-thioredoxin complex (PDB ID: 2O8V) (d), the TXNIP-thioredoxin complex (PDB ID: 4LL1) (e), the NLRP1-thioredoxin complex (PDB ID: 7WGE) (f), and the T7 DNA polymerase-thioredoxin-primer/template DNA complex (PDB ID: 6N7W) (g). The thioredoxins are shown in orchid.

Source data

Extended Data Fig. 4 The domain characteristics of Cas12p.

a, Atomic model fitted into the cryo-EM density map of Cas12p-TrxA-sgRNA-target DNA (29-nt TS and 11-nt NTS) complex. b, Domain structures of Cas12p. α helices and β strands in the WED and RuvC domains are numbered in orange and blue, respectively. c, Comparative sequence alignment of TB domains across the Cas12p family, demonstrating the hydrophobic amino acids enrichment within TB.a and the prevalence of positively charged residues within TB.b. The hydrophobic and basic amino acids are shown in grey and purple background, respectively. d, In vitro DNA cleavage assay using three RuvC domain mutants (D378A, E478A, and D582A). Each mutation individually results in undetected cleavage activity.

Source data

Extended Data Fig. 5 Characterization of the interaction between TrxA and Cas12p.

a, Illustration of the hydrophobic pocket formed by TB and REC domains. b, SDS-PAGE results for supernatants of wild-type Cas12p purified with wild-type (WT) or mutant His-TrxA, visualized with CBB staining. c, Evaluation of the functionality of wild-type (WT) and trxA mutants in an E. coli trxA-deletion strain utilizing bacterial spot assays, with non-targeting (NT) or targeting (T) sgRNA. d, SDS-PAGE results for supernatants of wild-type (WT) or mutant Cas12p proteins purified with wild-type His-TrxA, visualized with CBB staining. e, Functional assessments of wild-type (WT) and mutant Cas12p proteins in E. coli wild-type strain using bacterial spot assays with non-targeting (NT) or targeting (T) sgRNA. f, Cryo-EM density map of key residues in the interaction interface of TB and TrxA (top), and TrxA C33-C36 disulfide bond and Cas12p C127 residue (bottom). g,h, Analysis of intermolecular disulfide bond formation in wild-type Cas12p with wild-type (WT) or mutant His-TrxA (C33S, C36S), as well as Cas12p mutant (C127S) with wild-type His-TrxA using non-reducing (g) and reducing (h) SDS-PAGE, visualized with CBB staining. i, A proposed model illustrating the assembly process between Cas12p and TrxA protein, emphasizing the transformation of intermolecular to intramolecular disulfide bonds.

Source data

Extended Data Fig. 6 The essential role of TrxA in modulating Cas12p’s activity.

a, Top-ranking model of Cas12p predicted by AlphaFold. The structural model was coloured by pLDDT scores. b, In vitro cleavage assay for Cas12p RNP complex purified from E. coli BL21(DE3) ΔtrxA with serially increasing the concentration of purified TrxA protein. The concentration of Cas12p RNP is 300 nM. c,d, Bacterial spot assays for Cas12p targeting Klebsiella pneumoniae 1.6366 (c) and Acinetobacter baumannii ATCC 17978 (d) strains, with a non-targeting (NT) or targeting (T) sgRNA. e, Schematic for phage plaque assay using CRISPR-Cas12p. f, Phage plaque assays of T4 and T7 phages on E. coli BL21(DE3) harboring CRISPR-Cas12p with non-targeting (NT) or targeting (T) sgRNA. Results for plaque-forming unit (PFU) per milliliter are shown as mean ± s.d. of three biological replicates. g, T4 phage plaque assays on E. coli BL21(DE3) trxA-deletion strain harboring CRISPR-Cas12p with (+trxA) or without trxA complementation. PFU/mL data are displayed as mean ± s.d. of three biological replicates. h, T7 phage plaque assays on E. coli BL21 (DE3) trxA-deletion strain containing the CRISPR-Cas12p system with a non-targeting (NT) or targeting (T) sgRNA.

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Extended Data Fig. 7 Construction and infection of T4Cas12p recombinant phage.

a, Plates showing T4 phage plaques on E. coli harboring pCas9phage and either pSGphage‑HR‑NT (non‑targeting; negative control) or pSGphage‑HR‑T (targeting; experimental group). b, PCR-based verification of T4Cas12p recombinant phage. T4WT band represents the wild-type T4 phage, and T4Cas12p band represents a recombinant T4 phage carrying Cas12p. c, Phage plaque assays of T4WT and T4Cas12p phages on wild-type (WT) E. coli BL21(DE3).

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Extended Data Fig. 8 Cryo-EM data analysis of the Cas12p-TrxA-sgRNA-target DNA (33-bp dsDNA) complex.

a, SDS-PAGE (stained with CBB, left) and Urea-PAGE (right) analysis of the Cas12p-sgRNA-target DNA (33-bp dsDNA) complex. b, Cryo-EM data processing workflow for the Cas12p-TrxA-sgRNA-target DNA (33-bp dsDNA) complex. c, Representative cryo-EM micrograph from a total of 8004 micrographs. d, Representative 2D class averages. e, The half-map Fourier shell correlation (FSC) curves. f, Direction distribution plot. g, Cryo-EM density map coloured by local resolution.

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Extended Data Fig. 9 The mechanism of Cas12p’s TB domain.

a, Cryo-EM density map and atomic model of the Cas12p-TrxA-sgRNA-target DNA (33-bp dsDNA) complex. b, Close-up view of REC domain, TB domain, and TrxA. c, Close-up view of TB.b domain, TrxA and sgRNA-DNA heteroduplex. d, Close-up view of the TB.b domain of Cas12p. Labels for positively charged residues are in blue, and labels for hydrophobic residues are in yellow. e, Structure of T7 DNA polymerase in complex with primer-template DNA duplex. Lysine and arginine residues are indicated in blue. f, Close-up view of the TB domain of T7 DNA polymerase. Labels for positively charged residues are in blue, and labels for hydrophobic residues are in yellow. g, Close-up view of the TB.b-interacting surface of TrxA, which is enriched in hydrophobic residues. h, Comparative in vitro DNA cleavage assays for Cas12p with wild-type (WT) and mutant TB.b domain. Single mutation (K137A, R138A, K149A, K150A, K156A, K159A, or K162A) revealed unaltered cleavage activity compared with the wild-type Cas12p.

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Supplementary information

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Wang, Z., Wang, Y., Gao, H. et al. Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA. Nat Microbiol 11, 81–93 (2026). https://doi.org/10.1038/s41564-025-02224-z

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