Abstract
Cas9 nucleases recognize and cleave their target DNA through base pairing of a guide RNA with a spacer adjacent to a protospacer adjacent motif (PAM). Streptococcus thermophilus Cas9 (St1Cas9), a smaller Cas9 orthologue than Streptococcus pyogenes Cas9, enables robust genome editing in diverse organisms. Here we report high-resolution structures of St1Cas9 in complex with a single-guide RNA and different PAM-containing DNAs. All of the structures represent an HNH catalytic state that is rarely observed in other Cas9 structures, clearly depicting the active conformation. A unique wing region in the REC domain forms intensive interactions with the HNH domain, playing a key role in regulating St1Cas9 DNA cleavage activity and probably stabilizing the active conformation. Furthermore, St1Cas9 applies a strategy distinct from those of other Cas9 orthologues for PAM recognition. Structure-guided engineering of St1Cas9 substantially expanded its targeting scope. These molecular-level characterizations will facilitate the rational engineering of St1Cas9.
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Data availability
Conservation analysis of the active-site residues of the HNH domains was performed from the Cas9 protein family (InterPro accession: IPR028629). The structures of St1Cas9 in complex with different PAMs have been deposited in the Protein Data Bank under the accession codes: St1Cas9/GGAA (6M0V), St1Cas9/AGAA (6M0W) and St1Cas9/AGGA (6M0X). The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank staff from BL19U1 beamlines of the National Facility for Protein Science Shanghai at Shanghai Synchrotron Radiation Facility for help with data collection. This work was financially supported by the National Natural Science Foundation of China (grant nos. 21922705, 91753127 and 31700123), National Key R&D Program of China Grant (no. 2017YFA0506800) and the Shanghai Science and Technology Committee Rising-Star Program (grant no. 19QA1406000) to Q.J.
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Contributions
Q.J. conceived and directed the project. Y.Z. and Q.J. designed the research. Y.Z. performed most of the experiments. H.Z., Z.W. and N.T. helped express and purify St1Cas9 proteins. H.Z., Yujue Wang, W.C., Yannan Wang and Yu Wang helped perform the cytosine base editing assay in K. pneumoniae. H.Z., Yujue Wang, W.C., Yannan Wang, Z.W., N.T. and Yu Wang helped with other experiments. X.X. and S.Z. performed the bioinformatics analysis. Y.Z. and Q.J. analysed the data. Y.Z., J.G. and Q.J. carried out crystallographic studies. Y.Z. and Q.J. wrote the paper.
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Extended data
Extended Data Fig. 1 Bacterial survival assay of St1Cas9 with full-length or truncated sgRNA.
a, Scheme of St1Cas9-mediated bacterial survival assay in Klebsiella pneumoniae cells. b, The dhaB3 spacer-introduced pCasKP-St1Cas9 plasmids containing the full-length sgRNA (middle) or the sgRNA with the deletion of stem loop2 (right) efficiently killed K. pneumoniae cells. The empty pCasKP-St1Cas9 was transformed into K. pneumoniae cells as a control (left).
Extended Data Fig. 2 The unique wing region is only present in St1Cas9 among the currently determined Cas9 structures.
a, The REC lobe of St1Cas9 in this study. The wing region is highlighted by the pink cycle. b–g, The REC lobes of SpCas9 (b), FnCas9 (c), SaCas9 (d), Nm1Cas9 (e), CdCas9 (f) and CjCas9 (g), respectively.
Extended Data Fig. 3 Interactions between St1Cas9 and the nucleic acids.
St1Cas9 residues that interact with the sgRNA-dsDNA duplex via their main chains are shown in parentheses. Water-mediated hydrogen-bonding interactions are not shown.
Extended Data Fig. 4 Distinct conformations of the Cas9 orthologs.
a, Post-catalytic state of DNA-bound SpCas9. Zoom-in view of the active sites of the SpCas9 HNH domain is shown in the right figure. b and c, Inactive states of DNA-bound SaCas9 (b) and DNA-bound FnCas9 (c). d, Catalytic state of DNA-bound Nm1Cas9. Zoom-in view of the active sites of the Nm1Cas9 HNH domain is shown in the left figure. e, Post-catalytic state of DNA-bound Nm1Cas9. f, Catalytic state of DNA-bound St1Cas9. All distances were measured between the Cα atom of the catalytic residue H and the scissile phosphate of TS nucleotides 3 and 4 in the catalytic or inactive state Cas9 structures (b–d, f), or between the Cα atom of the catalytic residue H and the 5’-PO3 of TS nucleotide 4 in the post-catalytic state Cas9 structures (a, e). The positions of the catalytic residue H were shown as yellow stars.
Extended Data Fig. 5 Catalytic pockets of the HNH domains.
a–c, Close-up view of Mg2+-binding catalytic pockets of the HNH domains of St1Cas9 (a), Nm1Cas9 (b) and AnCas9 (c). Mg2+ and water molecules were coloured green and red, respectively. d, In vitro DNA cleavage assay of the wild type and various mutants of St1Cas9. The linearized plasmid containing a target sequence was used as the substrate and the full-length sgRNA was used as the guide RNA in the assay.
Extended Data Fig. 6 DNA binding activities of the wild type St1Cas9 and the variant with the truncation of the wing region.
Electrophoretic mobility shift assay was performed using catalytic inactive dCas9-sgRNA complexes and the FAM-labelled target dsDNA.
Extended Data Fig. 7 Phylogenetic tree of putative wing region containing Cas9s.
Each sequence represents sequences with >70% sequence identity.
Extended Data Fig. 8 In vitro cleavage activities of wild type St1Cas9 and the variant with the truncation of the wing region.
a, The spacer sequences of the linearized plasmids with single or double mismatches at positions 20-16. b, In vitro DNA cleavage assay of wild type St1Cas9. The linearized plasmids containing double mismatches at positions 20-16 were used as the substrates. c, In vitro DNA cleavage assay of wild type St1Cas9. The linearized plasmids containing single mismatches at positions 20-16 were used as the substrates. d, In vitro DNA cleavage assay of wild type St1Cas9 and the variant with the truncation of the wing region. The linearized plasmids containing single mismatches at positions 20 or 19 were used as the substrates. The reaction condition of wild-type St1Cas9 was 5 nM linearized plasmid, 120 nM Cas9, and 240 nM sgRNA. The reaction condition of the St1Cas9 variant with the truncation of the wing region was 5 nM linearized plasmid, 500 nM Cas9, and 1 µM sgRNA.
Extended Data Fig. 9 In vitro cleavage activities of the wild type and various mutants of St1Cas9.
a, In vitro DNA cleavage assay of the wild type and various K1086 mutants of St1Cas9. The linearized plasmids containing the 5’-AAANAA-3’ PAM sequences were used as the substrates. b, In vitro DNA cleavage assay of the wild type and K1086L mutant of St1Cas9. The linearized plasmids containing the 5’-AANTAA-3’ PAM sequences were used as the substrates. c, In vitro DNA cleavage assay of the wild type and various engineered mutants of St1Cas9. The linearized plasmids containing the 5’-AARTAA-3’ PAM sequences were used as the substrates.
Extended Data Fig. 10 Determination of the editing window of St1Cas9-mediated cytosine base editing system in K. pneumoniae.
a, Scheme of St1Cas9-mediated cytosine base editing. b, The editing results of various C-rich spacers in the KP_1.6366 strain. The Cs were coloured red and the target PAM sites were coloured blue. The experiment was performed in triplicate. Error bars report standard deviations of the mean (s.d.).
Supplementary information
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Supplementary Figs. 1–4 and Tables 1–4.
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Base editing in K. pneumoniae.
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Zhang, Y., Zhang, H., Xu, X. et al. Catalytic-state structure and engineering of Streptococcus thermophilus Cas9. Nat Catal 3, 813–823 (2020). https://doi.org/10.1038/s41929-020-00506-9
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DOI: https://doi.org/10.1038/s41929-020-00506-9
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