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Structural visualization of the molecular evolution of CRISPR–Cas9

Nature Structural & Molecular Biology (2026)Cite this article

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Abstract

RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR–Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700–1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases—two IscBs and two Cas9s—each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR–Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.

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Fig. 1: Phylogenetic distribution of IscB and Cas9.
Fig. 2: Cryo-EM structures of HfmIscB, TbaIscB, YnpsCas9 and NbaCas9.
Fig. 3: IscB and Cas9 structures.
Fig. 4: Guide RNA architectures.
Fig. 5: Coevolution of the nucleases and their RNA guides.
Fig. 6: Target DNA recognition and cleavage.
Fig. 7: Molecular evolution of CRISPR–Cas9.

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Data availability

The structural models were deposited to the Protein Data Bank under accession codes 9K2Z (HfmIscB), 9K30 (TbaIscB), 9K31 (YnpsCas9) and 9K32 (NbaCas9). The cryo-EM density maps were deposited to the EM Data Bank under accession codes EMD-62001 (HfmIscB), EMD-62002 (TbaIscB), EMD-62003 (YnpsCas9) and EMD-62004 (NbaCas9). The raw images were deposited to the EM Public Image Archive under accession code EMPIAR-12424. All other data supporting the 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 K. Makarova for assistance with the phylogenetic analysis and staff scientists at The University of Tokyo’s cryo-EM facility, especially Y. Sakamaki, for help with cryo-EM data collection. N.N. is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25KJ1023. K.K. was supported by a grant from the Mitsubishi Foundation. F.Z. is supported by National Institutes of Health grants (1DP1-HL141201 and 2R01HG009761-05), the Howard Hughes Medical Institute, Open Philanthropy, the Edward Mallinckrodt, Jr. Foundation, the Poitras Center for Psychiatric Disorders Research at MIT, the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Yang-Tan Molecular Therapeutics Center at McGovern, the Phillips family and J. and P. Poitras. H.N. is supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from the Japan Agency for Medical Research and Development under grant number JP21am0101115 (support number 2792), JSPS KAKENHI grant numbers 21H05281 and 25H00436, the Takeda Medical Research Foundation and the Inamori Research Institute for Science.

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Author notes

  1. These authors contributed equally: Naoto Nagahata, Kazuki Kato, Sota Yamada.

Authors and Affiliations

  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

    Naoto Nagahata, Sota Yamada, Masahiro Hiraizumi & Hiroshi Nishimasu

  2. Mechanistic Immunology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan

    Kazuki Kato & Yukari Isayama

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Soumya Kannan & Feng Zhang

  4. McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan & Feng Zhang

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan & Feng Zhang

  6. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan & Feng Zhang

  7. Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan

    Sae Okazaki, Keitaro Yamashita & Hiroshi Nishimasu

  8. Computational Biology Branch, Division of Intramural Research, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

    Eugene V. Koonin

  9. Howard Hughes Medical Institute, Cambridge, MA, USA

    Feng Zhang

  10. Inamori Research Institute for Science, Kyoto, Japan

    Hiroshi Nishimasu

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Contributions

N.N., K.K. and S.Y., with assistance from S.O. and Y.I., performed the biochemical experiments. N.N., K.K., S.Y., M.H., K.Y. and H.N. performed the structural analyses. S.K., with assistance from F.Z., performed the TAM/PAM screening experiments. N.N. and H.N., with assistance from S.Y., K.K., S.K., M.H., K.Y. and E.V.K., wrote the manuscript. H.N. supervised the research.

Corresponding author

Correspondence to Hiroshi Nishimasu.

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Competing interests

F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies and Proof Diagnostics. The remaining authors declare no competing interests.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Melina Casadio, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 TAM/PAM specificities of IscBs and Cas9s.

(a) Weblogo motifs of TAMs for HfmIscB and TbaIscB and PAMs for YnpsCas9 and NbaCas9. (b–e) TAM/PAM recognition by HfmIscB (b), TbaIscB (c), YnpsCas9 (d), and NbaCas9 (e). Schematics and structures are shown on the top and bottom, respectively. Cryo-EM density maps for TAM/PAM-interacting residues and the DNA molecules are shown as gray semitransparent surfaces. Disordered nucleotides are indicated by dotted lines in (b). In the HfmIscB structure, dG3*, dA4*, dG5*, and dG6* are recognized by K388, N379, Y372, and K389 through base-specific hydrogen-bonding interactions, respectively. Although our biochemical data indicate the preference for the second G nucleotide in the TAM, the dG2* nucleobase does not directly contact the HfmIscB protein, suggesting that dG2* is recognized by nearby residues, such as H380 and Q381, via water-mediated hydrogen bonds. In the TbaIscB structure, dG1* and dG2* are recognized by Q504 and N416, respectively. It is likely that G at position 3* in the PAM is recognized by K497. While YnpsCas9 recognizes dC−2 and dG3* using K580 and K642, respectively, NbaCas9 contacts dG2* and dC−2 using N638/R722 and K636, respectively.

Extended Data Fig. 2 Multiple sequence alignment.

Multiple sequence alignment of HfmIscB, TbaIscB, YnpsCas9, and NbaCas9. HfmIscB, IscB from the human fecal metagenome (Ga0169696_100022); TbaIscB, IscB from Tissierellia bacterium (JAAZKS010000250.1); YnpsCas9, Cas9 from the sediment metagenome in Yellowstone National Park (Ga0315277_10040887); NbaCas9, Cas9 from Nitrospirae bacterium (MHDT01000042.1). The figure was prepared using Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) and ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript).

Extended Data Fig. 3 Structural comparison of the IscB and Cas9 orthologs.

Structures of the nuclease–guide RNA–target DNA complexes for HfmIscB, TbaIscB, OgeuIscB (PDB: 7XHT), YnpsCas9, NbaCas9, Acidothermus cellulolyticus Cas9 (AcCas9) (PDB: 8D2L), Campylobacter jejuni Cas9 (CjCas9) (PDB: 5X2G), Francisella novicida Cas9 (FnCas9) (PDB: 5B2O), and Streptococcus pyogenes Cas9 (SpCas9) (PDB: 5F9R).

Extended Data Fig. 4 Structural comparison of the REC regions.

(a) Structural comparison of the REC3 domains of TbaIscB, YnpsCas9, NbaCas9, AcCas9 (PDB: 8D2L), FnCas9 (PDB: 5B2O), and SpCas9 (PDB: 5F9R). Zinc ions are shown as gray spheres. The zinc-coordinating residues are shown as stick models. Disordered residues are indicated by dotted lines. (b) Guide–target heteroduplex recognition by the REC regions of HfmIscB, TbaIscB, OgeuIscB (PDB: 7XHT), YnpsCas9, NbaCas9, AcCas9 (PDB: 8D2L), CjCas9 (PDB: 5X2G), FnCas9 (PDB: 5B2O), and SpCas9 (PDB: 5F9R). βHI, β-hairpin insertion. The α-helices comprising the helix bundles are numbered.

Extended Data Fig. 5 Sequence and structural diversity of IscB-L.

(a) Maximum likelihood phylogenetic tree of IscB-L. The nine clades of IscB-L are highlighted in different colors, with selected orthologs indicated by dots. (b) Cryo-EM structure of OgeuIscB (PDB: 7XHT) and predicted models of selected IscB-L orthologs. The models were predicted by Boltz-1. The PLMP, HNH, WED, and TI domains and the L1/L2 linkers are shown as semitransparent ribbon models for clarity.

Source data

Extended Data Fig. 6 Recognition of the first stem, TAM/PAM-proximal heteroduplex, and central stem.

(a–d) Recognition of the first stems (the guide adaptor stem for HfmIscB and the repeat:antirepeat duplex for TbaIscB and Cas9s), the TAM/PAM-proximal heteroduplex, and the central stem by the HfmIscB (a), TbaIscB (b), YnpsCas9 (c), and NbaCas9 (d) complexes. The residues and nucleotides forming hydrogen bonding and stacking interactions are depicted as stick models. Hydrogen bonds are indicated by dashed lines. SL, stem loop; GAS, guide adaptor stem; R:AR, repeat:antirepeat; RH, recognition hairpin; RS, recognition stem; CS, central stem; NS, nexus stem.

Extended Data Fig. 7 RuvC and HNH active sites of the Cas9 nucleases.

(a) Structural comparison of the DNA targets bound to the RuvC and HNH active sites between YnpsCas9, NbaCas9, and SpCas9 (PDB: 7Z4J). The active residues of the RuvC and HNH domains are shown as space-filling models. Disordered regions are indicated by dotted lines. TS, target DNA strand; NTS, nontarget DNA strand. (b) Recognition of the NTS in the RuvC active sites of YnpsCas9, NbaCas9, and SpCas9. Magnesium ions are shown as gray spheres. Hydrogen and coordinate bonds are indicated by dashed lines. (c) Recognition of the TS in the HNH active sites of YnpsCas9, NbaCas9, and SpCas9. Magnesium ions and water molecules are shown as gray and red spheres, respectively. Hydrogen and coordinate bonds are indicated by dashed lines.

Extended Data Fig. 8 Nonspecific single-stranded RNA cleavage by TbaIscB.

(a) In vitro RNA and DNA cleavage activities of WT TbaIscB and its active-site mutants. The TbaIscB protein (WT, dRuvC, or dHNH) was incubated with either the Cy5-labeled single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or ssDNA substrate (60 nt) at 37 °C for 1 or 5 min, and then the reaction was analyzed by 15% urea-PAGE. dRuvC, D59A; dHNH, H256A. (b) In vitro ssRNA cleavage activities of TbaIscB, HfmIscB, YnpsCas9, and NbaCas9. The Cy5-labeled ssRNA substrate (60 nt) was incubated with either the TbaIscB, YnpsCas9, or NbaCas9 protein, or the HfmIscB–ωRNA complex at 37 °C for 1 or 60 min, and then the reaction was analyzed by 15% urea-PAGE. HfmIscB was only purified as the ribonucleoprotein complex. In (a) and (b), experiments were repeated three times with similar results. (c) Multiple sequence alignment of the HNH domains from TbaIscB, NmCas9, CjCas9, OgeuIscB, HfmIscB, YnpsCas9, and NbaCas9. Key residues for RNA cleavage are highlighted in red. (d) Structural comparison of the HNH domains of TbaIscB (AlphaFold2 model) and NmCas9 (PDB: 8JA0).

Source data

Extended Data Fig. 9 Structural comparison of the active states of Cas9s.

(a) Structural comparison of the active states of YnpsCas9, NbaCas9, AcCas9 (PDB: 8D2L), and SpCas9 (PDB: 7Z4J). The HNH domains and the L1/L2 linkers are shown as ribbon models, while the rest of the complex is shown as surface models. (b, c) Interactions of the HNH domain with the REC linker and sgRNA (b) and with the WED domain (c) in the YnpsCas9 active state. (d and e) Interactions of the HNH domain with the REC1 domain (d) and with the WED domain (e) in the NbaCas9 active state. (f) Comparison of the spatial arrangements between the RuvC/HNH domains and the L1/L2 linkers in YnpsCas9, NbaCas9, AcCas9 (PDB: 8D2L), and SpCas9 (PDB: 7Z4J). The residues interacting with NTS nucleobases are shown as space-filling models.

Extended Data Fig. 10 Sequence and structural diversity of IsrB, IscB-S, IscB-L, and type II-D Cas9.

(a) Maximum likelihood phylogenetic tree of IsrB, IscB, and type II-D Cas9, built based on their RuvC domains and bridge helices. The branches of IsrB, IscB-S, IscB-L, and type II-D Cas9 are highlighted in different colors. The CRISPR-associated and non-CRISPR-associated orthologs are indicated by gray and white dots, respectively. (b) Cryo-EM structures of DtIsrB (PDB: 8DMB), OgeuIscB (PDB: 7XHT), HfmIscB, TbaIscB, YnpsCas9, NbaCas9 and predicted models of selected IscBs (55826, 6734, 13572, 40754, 26022, and 48100). The models were predicted by Boltz-1. The PLMP, HNH, WED, and TI/PI domains and the L1/L2 linkers are shown as semitransparent ribbon models for clarity. The zinc ion and the zinc-coordinating residues are shown as space-filling models. ZF, zinc finger.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–13.

Reporting Summary

Peer Review File

Supplementary Table 1

DNAs and RNAs used in this study.

Supplementary Video 1

Cryo-EM structure of the HfmIscB–ωRNA–target DNA complex.

Supplementary Video 2

Cryo-EM structure of the TbaIscB–ωRNA–target DNA complex.

Supplementary Video 3

Cryo-EM structure of the YnpsCas9–guide RNA–target DNA complex.

Supplementary Video 4

Cryo-EM structure of the NbaCas9–guide RNA–target DNA complex.

Supplementary Video 5

A 3D variability analysis of the HfmIscB–ωRNA–target DNA complex.

Supplementary Video 6

A 3D variability analysis of the TbaIscB–ωRNA–target DNA complex.

Supplementary Video 7

A 3D variability analysis of the YnpsCas9–guide RNA–target DNA complex.

Supplementary Video 8

A 3D variability analysis of the NbaCas9–guide RNA–target DNA complex.

Source data

Source Data Fig. 1 and Extended Data Figs. 5 and 10

Cluster and sequence information and tree files used in the phylogenetic analyses.

Source Data Figs. 5 and 6 and Extended Data Fig. 8

Uncropped gel images.

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Nagahata, N., Kato, K., Yamada, S. et al. Structural visualization of the molecular evolution of CRISPR–Cas9. Nat Struct Mol Biol (2026). https://doi.org/10.1038/s41594-025-01743-x

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