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Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity

Nature volume 630, pages 484–492 (2024)Cite this article

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Abstract

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel β-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR–Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9–PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and ‘growth evolutionary’ trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs—such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

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Fig. 1: Large II-C Cas9s are associated with neighbourhood NAGs and exhibit structural growth trajectories.
Fig. 2: Cryo-EM structures of CbCas9 ternary and CbCas9–PcrIIC1 quaternary complexes.
Fig. 3: PcrIIC1 enhances the in vitro DNA interference activity of CbCas9.
Fig. 4: The CbCas9–PcrIIC1 complex enhances dsDNA-screening and dsDNA-invading capacities through two CbCas9 effectors.
Fig. 5: The CbCas9–PcrIIC1 complex boosts bacterial immunity against phages.

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Data availability

The electron density maps have been deposited to the Electron Microscopy Data Bank under the accession numbers EMD-37652, EMD-35827, EMD-37656, EMD-37762 and EMD-37657. The atomic coordinates and structure factors have been deposited to the Protein Data Bank under the accession numbers 8WMH, 8IYQ, 8WMM, 8WR4 and 8WMN. The genomic sequences of identified Cas9s in the HBGC dataset are provided in Supplementary Data 1. The next-generation sequencing dataset containing RNA-seq raw reads is available on the Sequence Read Archive under BioProject PRJNA1096841. Uncropped gels are provided in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

EM data were collected at the Tsinghua Cryo-EM facility. The data were analysed using the Bio-Computation platform at the Tsinghua University Branch of the Chinese National Center for Protein Sciences (Beijing). We thank J. F. Xu and X. Y. Cui for assistance with cryo-EM sample preparation; J. L. Lei, X. M. Li, F. Yang and N. Y. Zhou for assistance with electron microscopy; J. B. Jiang and M. Tian for assistance with protein purification; X. Ke and Z. A. Hu for providing ssRNA substrates; P. Q. Wang for help with language; and the Tsinghua University Technology Center for Protein Research, Genome Sequencing and Analysis at Tsinghua University for support. The cartoons in Figs. 2a, 3c–e, 4f,g and 5c, Extended Data Figs. 1a, 4c, 5d, 7a, 8a, 10a,d and Supplementary Fig. 9a were created with BioRender.com. Support for this project came from the National Key R&D Program (2022YFF1002801 to J.-J.G.L. and 2022YEF1001800 to Y.B.); the National Natural Science Foundation of China (32150018 to J.-J.G.L., 32101195 to S.Z. and 22061160466 and 22277063 to C.C.); the STI2030-Major Projects 2022ZD0213900 (to J.-J.G.L); the Ministry of Agriculture and Rural Affairs of China (J.-J.G.L. and C.G.); start-up funds from Tsinghua University (J.-J.G.L.); the Strategic Priority Research Program and Young Scientists in Basis Research in the Chinese Academy of Sciences (XDA24020104 and YSBR-078 to Y.B.); the New Cornerstone Science Foundation through the XPLORER PRIZE (Y.B.); the Hainan Excellent Talent Team (Y.B.); and the China Postdoctoral Science Foundation, general funding (2022M711848 to S.Z.).

Author information

Author notes

  1. These authors contributed equally: Shouyue Zhang, Ao Sun, Jing-Mei Qian, Shuo Lin

Authors and Affiliations

  1. Beijing Frontier Research Center for Biological Structure, Beijing Advanced Innovation Center for Structural Biology, State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Shouyue Zhang, Ao Sun, Shuo Lin, Wenjing Xing, Yun Yang, Han-Zhou Zhu, Yun Liu, Yu Meng, Shu-Lin Jin, Wenhao Song, Cheng-Ping Li, Zhaofu Li, Shuai Jin, Chunlai Chen & Jun-Jie Gogo Liu

  2. State Key Laboratory of Plant Genomics, CAS-JIC Centre of Excellence for Plant and Microbial Sciences, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Jing-Mei Qian, Xin-Yi Zhou, Yan-Shuo Guo & Yang Bai

  3. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Jing-Mei Qian, Xin-Yi Zhou, Yan-Shuo Guo, Shuai Jin, Caixia Gao & Yang Bai

  4. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Shuai Jin & Caixia Gao

  5. National Institute of Biological Sciences, Beijing, China

    Jian-Hua Wang & Meng-Qiu Dong

  6. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    Jian-Hua Wang & Meng-Qiu Dong

  7. Peking-Tsinghua Center for Life Sciences, College of Life Sciences, Peking University, Beijing, China

    Yang Bai

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  1. Shouyue Zhang
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  2. Ao Sun
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  3. Jing-Mei Qian
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Contributions

Project design: J.-J.G.L., Y.B., C.C., S.Z., C.G., A.S., J.-M.Q. and S.L. Bioinformatics: S.Z., Y.L., W.S. and C.-P.L. Biochemical analysis: A.S., W.X., Y.Y., Y.M., S.-L.J., Z.L., J.-H.W. and M.-Q.D. Structure analysis: S.Z., S.L. and H.-Z.Z. In vivo bacterial and anti-phage analysis: J.-M.Q., A.S., X.-Y.Z., Y.-S.G. and Y.M. Writing: J.-J.G.L., S.Z., A.S., J.-M.Q., S.L., S.J., Y.B. and C.C. Project supervision: J.-J.G.L.

Corresponding authors

Correspondence to Chunlai Chen, Yang Bai or Jun-Jie Gogo Liu.

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Competing interests

J.-J.G.L., S.Z., A.S. and S.L. are co-inventors on a patent application (CNIPA application 202410263253.1) related to this work, filed by Tsinghua University. This patent is entitled ‘A system for gene editing and/or phage resistance and its application’ and covers nucleic acid manipulation applications using CbCas9 and/or PcrIIC1. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Bioinformatic identification and conservation of II-C Cas9s.

a, Pipeline of identifying novel Cas9 systems from self-collected dataset. b, Length distribution of Cas9s in HBGC dataset. NAG+ Cas9s are denoted as squares in various colours and NAG− Cas9s are denoted as circles in blue. Dashed line marks the size of the smallest NAG+ Cas9 with 1,400 aa. c, Box plot illustrating protein size variation of Cas9s within different types in GEM/HBGC and Makarova NCBI datasets. Protein integrity within each dataset was manually checked with AlphaFold prediction. The number of Cas9s within each group is labelled above each box. The box plots show the minima, maxima, centre, bounds of box and whiskers and percentile with all data points. d, Primary sequence conservative analysis of II-C Cas9s. Identified II-C Cas9s were aligned to CjCas9 to calculate the conservation ratio of each amino acid. Amino acids of CjCas9 with conservation ratio above 0.98 are labelled. Specifically, catalytic amino acids of HNH and RuvC are coloured in red and blue, respectively. They are D8, E479, and D710 in the RuvC domain and H559, N573, and N582 in the HNH domain referenced by CjCas9. The red dashed line marks 0.98 (high conservation) and the black dashed line marks 0.2 (low conservation). e, Left, structural alignment of full-length Cas9 proteins, indicating HNH domains exhibit highly flexible orientation. Right, structural alignment of extracted stand-alone HNH domains, indicating a conserved structure of HNH domains in Cas9 proteins.

Extended Data Fig. 2 Structural evolutionary trajectory analysis of II-C Cas9s.

a, SGT analysis plane maps to emerging structural features. The size of the point corresponds to Cas9 size and the colour corresponds to different structural features. b, Schematic illustrating three evolutionary trajectories and representative II-C Cas9 models at checkpoint of each evolutionary trajectory. The emerging domains are coloured and labelled according to a. c, SGT analysis plane maps to NAG association. The size of points corresponds to Cas9 size and the colour corresponds to different NAG association.

Extended Data Fig. 3 Phylogenetic analysis of II-C Cas9s and ribosomal proteins.

a, Phylogenetic analysis of II-C Cas9s. The outgroup includes three IsrB and three IscB proteins. NAGs are coloured according to Supplementary Fig. 2. SGT clusters are marked with various colours on the second outermost ring. Protein size is shown on the outermost ring. b, Phylogenetic tree of ribosomal proteins from Cas9-containing genomes. The phylogenetic tree was built as previously described by concatenating 16 ribosomal proteins (L2, L3, L4, L5, L6, L14, L15, L16, L18, L22, L24, S3, S8, S10, S17 and S19). Phylum is coloured according to the legend. Fusobacteriota, which locate near the root of bacteria phylogeny tree is coloured red. Protein size is shown on the outermost ring.

Extended Data Fig. 4 Cas9-associated PcrIIC1 is a non-toxic protein with 3′–5′ ssDNA nuclease activity.

a, RNA-seq analysis of gene locus of PIN-associated CRISPR–Cas9 system in Chryseobacterium sp. by PE150 (blue) and SE50 (red) strategies showing the expression of Cas9, PIN, Cas1, Cas2 and RNA components (tracrRNA and CRISPR array). b, Phylogenic analysis of Cas9-associated PIN (green) with other PIN-domain toxin proteins (light blue) and Doc toxin proteins (yellow; outgroups). c, Left, model of in vivo toxin assay in E. coli BW25141 revealed by monitoring optical density (OD) over time. Right, growth curve of E. coli BW25141 containing different plasmids expressing MBP, SUMO, PcrIIC1 or VapC toxin with 1% glucose repressor (top) or 25 mM arabinose inducer (bottom). OD600 was monitored over time. Data are presented as mean ± s.d. (n = 3 biologically independent wells). d, In vitro cleavage of 3′- or 5′-Fam-labelled ssDNA targets by PcrIIC1 revealed by denaturing PAGE, S means substrates and P means cleavage products. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). e, In vitro cleavage of two different Fam-labelled dsDNA targets by PcrIIC1 revealed by denaturing PAGE. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). f, In vitro cleavage of three different 5′- or 3′-Fam-labelled ssRNA targets by PcrIIC1 revealed by denaturing PAGE. The gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). All aliquots were collected at the following time points: 0, 2, 5, 15, 30, 60, 90 and 120 min. For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 5 Biochemical properties of CbCas9.

a, Left, in vitro cleavage of dsDNA targets containing different PAMs by CbCas9 at 15 min revealed by denaturing PAGE, S denotes substrates and P means cleavage products. Right, bar plot illustrating the cleavage efficiency of CbCas9 on each dsDNA target (PAMs are shown on the X-axis). Gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). b, Cleavage site mapping of SpCas9 (left) and CbCas9 (right) on the NTS or TS revealed by denaturing PAGE. S means substrates, PNTS means cleavage products of non-target strand and PTS means cleavage products of target strand. Gel image is representative of four independent experiments with similar results (n = 4 independent replicates). DNA markers of 28-nt, 30-nt, 32-nt and 34-nt were labelled. c, Model depicting cleavage sites for NTS and TS by SpCas9 and CbCas9 (marked in red arrows, PAM in blue). d, Schematic representation of DNA target (6-nt complementary) recognition by CbCas9. The hydrogen bonds of K1365 with N7 of dA3 on NTS, and Q1397 with O4 of dT(−4) on TS are coloured in red. The interactions with DNA backbone are coloured in blue. e, Left, In vitro cleavage of NTS and TS by CbCas9 and β-REC2-mutated CbCas9 revealed by denaturing PAGE; aliquots were collected with time points: 0, 2, 5, 15, 30, 60, 90, and 120 min (n = 3, each). Right, in vitro NTS and TS cleavage efficiency plot of CbCas9 and β-REC2-mutated CbCas9 on dsDNA substrates. Gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). The rate constant k values for CbCas9 in NTS and TS are 0.2053 and 0.3100; for β-REC2-mutated CbCas9, the corresponding k values are 0.055 and 0.047, respectively). For gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 6 MST and cryo-EM analysis of the binding between CbCas9 and PcrIIC1 in three stages.

a, CbCas9–PcrIIC1 complex. Top: equilibrium dissociation constant (Kd) determined by MST, with 25 nM Red-tris-NTA labelled CbCas9 and varying concentrations of PcrIIC1. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. dCbCas9 indicates deactivated CbCas9 (D9A, H837A). Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins were fitted in the semitransparent map. b, CbCas9–PcrIIC1–sgRNA complex. Top: Kd determined by MST. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins and nucleic acids were fitted in the semitransparent map. c, CbCas9–PcrIIC1–sgRNA–dsDNA (28-bp) complex. Top: Kd determined by MST. Data are presented as mean ± s.d. (n = 3 independent replicates). Graphs are represented as FNorm [‰] against PcrIIC1 concentration. Mid: 2D class of particles. Bottom: cryo-EM map after Gaussian filtering (sDev 6) and coloured according to each compartment. Atomic models of proteins and nucleic acids were fitted in the semitransparent map.

Source Data

Extended Data Fig. 7 PcrIIC1 enhances the DNA-binding capability, PAM tolerance and target cleavage efficiency of CbCas9 by interacting with the CTH domain.

a, Top, model of in vivo bacterial genome interference assay for CbCas9 and PcrIIC1. Bottom, bacteria genome interference assay revealed by bacterial culture plate with a series of 10-fold dilution gradients. CbCas9 and CbCas9–PcrIIC1 groups with non-target spacer and target spacers are denoted as Cb and Cb+P separately (Different targeting PAMs are shown above the plate). Data are representative of three independent experiments with similar results (n = 3 biologically independent replicates). b, Efficiency plot of the DNA-binding capability of CbCas9 and CbCas9–PcrIIC1 on 62-bp dsDNA target with different PAM (ACAAA and ACAAC) evaluated by EMSA. Data are presented as mean ± s.d. from three independent experiments. dCb and dCb+P indicate deactivated CbCas9 (D9A, H837A) and deactivated CbCas9–PcrIIC1 effectors, respectively. c, Top, in vitro cleavage of dsDNA targets with different PAMs by CbCas9 and CbCas9–PcrIIC1 at 15 min, revealed by denaturing PAGE. S denotes substrates. Bottom, bar plot of cleavage efficiency (different PAMs are shown on the x axis). The gel shown is representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). CbCas9 and CbCas9–PcrIIC1 groups are represented by Cb and Cb+P separately. Statistical analysis was calculated using two-way ANOVA with multiple comparisons; ns, P > 0.1234; *P > 0.0332; **P > 0.0021; ***P > 0.0002; ****P < 0.0001. d, In vitro cleavage of dsDNA targets with ACAAA, ACAAC and ACGTC PAM by CbCas9 and CbCas9–PcrIIC1 revealed by denaturing PAGE; aliquots were collected at time points: 0, 2, 5, 15, 30, 60, 90, and 120 min. Bottom right, bar chart showing cleavage efficiency of different dsDNA targets at 120 min. Gels shown are representative of three independent experiments with similar results and data are presented as mean ± s.d. (n = 3 independent replicates). Statistical analysis was calculated using two-way ANOVA with multiple comparisons; ns, P > 0.1234; ****P < 0.0001. e, In vitro cleavage of target dsDNA by CbCas9 and CTH-mutated CbCas9 with or without PcrIIC1 revealed by denaturing PAGE. Gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). Cb and Cb+P indicate CbCas9 and CbCas9–PcrIIC1 effectors, respectively. Cb-CTH mut and Cb-CTH mut+P indicate CTH-mutated CbCas9 and CTH-mutated CbCas9–PcrIIC1 effectors, respectively. f, Top, nucleotide sequence of CRISPR repeats. The selected GTTGT PAM is labelled with a red line. Bottom, In vitro cleavage of target dsDNA with GTTGT PAM revealed by denaturing PAGE. The gel shown is representative of three independent experiments with similar results (n = 3 independent replicates). g, In vivo bacterial genome self-targeting assay revealed by bacterial culture plate with a series of tenfold dilution gradients. CbCas9 and CbCas9–PcrIIC1 groups with non-target spacer and target spacers are represented by Cb and Cb+P separately (Targeting PAM is shown above the plate). Data are representative of three independent experiments with similar results (n = 3 biologically independent replicates). For image and gel source data, see Supplementary Fig. 1.

Source Data

Extended Data Fig. 8 PcrIIC1 enhances CbCas9 interference activity on mismatched or long dsDNA targets.

a, Left, model depicting in vitro evaluation of the mismatch tolerance on the protospacer region by CbCas9 and CbCas9–PcrIIC1 effectors. Right, bar chart illustrating the mismatch tolerance ratio of CbCas9 and CbCas9–PcrIIC1 effectors at different positions. The mismatch tolerance ratio of 0 means that the cleavage products are perfectly paired with the sgRNA at that position, and ratio of 1 means 25% paired (randomly distribution; see Methods). Data are presented as mean ± s.d. from three independent experiments. Statistical analysis was calculated using two-sided test, with significance determined by t-test. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. b, In vitro cleavage assay of target dsDNA containing ACAAC PAM with different lengths (28, 62, 300 and 600 bp) revealed by denaturing PAGE (28- and 62-bp dsDNA targets) and agarose gel (300- and 600-bp dsDNA targets); aliquots were collected with time points: 0, 2, 5, 15, 30, 60, 90, and 120 min. Gels shown are representative of three independent experiments with similar results (n = 3 independent replicates). For gel source data, see Supplementary Fig. 1. c, Cleavage efficiency plot of target dsDNA with different lengths by CbCas9 and CbCas9–PcrIIC1 effectors. Data are presented as mean ± s.d. (n = 3 independent replicates). d, Bar chart showing cleavage efficiency of dsDNA targets with different lengths at 120 min by CbCas9 and CbCas9–PcrIIC1 effectors. Data are presented as mean ± s.d. (n = 3 independent replicates). Statistical analysis was calculated using two-way ANOVA with multiple comparisons; ns, P > 0.1234; ****P < 0.0001. e, Equilibrium dissociation constant (Kd) of PcrIIC1 with CbCas9–sgRNA–dsDNA(62bp) was evaluated using MST, with 25 nM Red-tris-NTA labelled CbCas9 and varying concentrations of PcrIIC1. Data are presented as mean ± s.d. from three independent experiments with consistent results. Graphs are represented as FNorm [‰] against PcrIIC1 concentration. dCbCas9 indicates deactivated CbCas9 (D9A, H837A).

Source Data

Extended Data Fig. 9 Structural details of the CbCas9–PcrIIC1 complex with long dsDNA.

a, Cryo-EM maps (left) and atomic models (right) of TS and sgRNA in the non-targeting complex. The Watson–Crick pairs are labelled in black and the wobble pairs are labelled in purple. b, Comparison of the DNA cryo-EM map in the CbCas9–PcrIIC1 non-targeting complex with a mimic B-form DNA model (yellow and green). The DNA region with a B-form helix was fitted using a mimic DNA backbone model (red and blue). c, Comparison of PAM interaction of preferred ACAAA PAM (left) and non-preferred CCAGA PAM (right). The hydrogen bonds are represented with blue dotted lines. d, Comparison of electrostatic interactions with the DNA by CbCas9 monomer and the CbCas9–PcrIIC1 non-targeting complex. e, Comparison of the DNA cryo-EM map in the CbCas9–PcrIIC1 dual-targeting complex with a mimic B-form DNA model (yellow and green). The DNA region with a B-form helix was fitted using a mimic DNA backbone model (red and blue).

Extended Data Fig. 10 PcrIIC1 enhances the anti-phage activity of CbCas9.

a, Model of the anti-phage assay performed with CbCas9 or CbCas9–PcrIIC1 effectors. b, Box plot showing in vivo anti-phage activity on Target-1 by CbCas9 (blue), CbCas9–PcrIIC1 (red), CTH-mutated CbCas9 (green) and CTH-mutated CbCas9–PcrIIC1 (yellow) effectors. Cb and Cb+P indicate CbCas9 and CbCas9–PcrIIC1 effectors, respectively; CTH mut and CTH mut+P indicate CTH-mutated CbCas9 and CTH-mutated CbCas9–PcrIIC1 effectors, respectively. All experimental data for each group were conducted in three batches, each with 18 replicates. The box plots show the minima, maxima, centre, bounds of box and whiskers and percentile with all data points. Statistical analysis was calculated using one-way ANOVA with multiple comparisons; ****P  <  0.0001. c, Box plot showing in vivo anti-phage activity with mismatched spacers on Target-1 by CbCas9 (yellow) and CbCas9+PcrIIC1 (green). Blue dashed line represents the median of lysis area for CbCas9 with perfectly matched spacer on Target-1. MM means mismatched and PM means perfectly matched. All experimental data for each group was conducted in three batches, each with 18 replicates. The box plots show the minima, maxima, centre, bounds of box and whiskers and percentile. 5 μl of T7 phage with a titre of 1010 was spotted. d, Schematic showing the design of dual targeting on T7 genome, mimicking the architecture of the dual-targeting dsDNA used in in vitro experiments. e, Top, box plot showing plaque areas of lysis zone to assess anti-phage ability by empty vector, CbCas9 (single-targeting), CbCas9 (dual targeting), CbCas9–PcrIIC1 (single-targeting), and CbCas9–PcrIIC1 (dual targeting). Bottom, representative images of each plaque. Cb and Cb+P indicate CbCas9 and CbCas9–PcrIIC1 effectors, respectively. All experimental data for each group were conducted in three batches, each with 18 replicates. Five microlitres of T7 phage with a titre of 109 was spotted. The box plots show the minima, maxima, centre, bounds of box and whiskers and percentile with all data points. Statistical analysis was calculated using one-way ANOVA with multiple comparisons; ****P  <  0.0001.

Extended Data Table 1 Statistics for cryo-EM analysis and model refinement
Full size table

Supplementary information

Supplementary Information

Supplementary Information containing Supplementary Figs. 1–21, legends for Supplementary Tables 1–4, legends for Supplementary Videos 1–3 and legends for Supplementary Data 1–6.

Reporting Summary

Peer Review File

Supplementary Table 1

CRISPR–Cas9 systems with NAG.

Supplementary Table 2

The II-C Cas9s in structural evolutionary trajectory analysis.

Supplementary Table 3

Structural quantification of the II-C Cas9s.

Supplementary Table 4

List of plasmids, RNA, DNA and strains used in this study.

Supplementary Data 1

Genomic sequences hosting identified Cas9s in the HBGC dataset.

Supplementary Data 2

Validation report of CbCas9 full R-loop ternary complex (8IYQ).

Supplementary Data 3

Validation report of CbCas9 partial R-loop ternary complex (8WMH).

Supplementary Data 4

Validation report of CbCas9–PcrIIC1 partial complex bound to 28-bp substrate (8WMM).

Supplementary Data 5

Validation report of CbCas9–PcrIIC1 non-targeting complex (8WR4).

Supplementary Data 6

Validation report of CbCas9–PcrIIC1 dual-targeting complex (8WMN).

Supplementary Video 1

Cas9 growth along trajectory 1. For each checkpoint along trajectory 1, five representative Cas9s are selected and aligned. The trajectory is shown at the top, with the checkpoint name displayed at the bottom left. Emerging domains are coloured and labelled according to Extended Data Fig. 2b. The size of each Cas9 is labelled at top left.

Supplementary Video 2

Cas9 growth along trajectory 2. For each checkpoint along trajectory 2, five representative Cas9s are selected and aligned. The trajectory is shown at the top, with the checkpoint name displayed at the bottom left. Emerging domains are coloured and labelled according to Extended Data Fig. 2b. The size of each Cas9 is labelled at top left.

Supplementary Video 3

Cas9 growth along trajectory 3. For each checkpoint along trajectory 3, five representative Cas9s are selected and aligned. The trajectory is shown at the top, with the checkpoint name displayed at the bottom left. Emerging domains are coloured and labelled according to Extended Data Fig. 2b. The size of each Cas9 is labelled at top left.

Source data

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Zhang, S., Sun, A., Qian, JM. et al. Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity. Nature 630, 484–492 (2024). https://doi.org/10.1038/s41586-024-07486-x

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