ABSTRACT CRISPR–Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and

single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from

Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts _Candidatus_ Altiarchaeum crystalense and _Ca_. A. horonobense, respectively, match

putative essential genes in their episymbionts’ genomes of the genus _Ca_. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals

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OTHERS UNDINARCHAEOTA ILLUMINATE DPANN PHYLOGENY AND THE IMPACT OF GENE TRANSFER ON ARCHAEAL EVOLUTION Article Open access 07 August 2020 METAGENOMIC CHARACTERIZATION OF VIRUSES AND MOBILE

GENETIC ELEMENTS ASSOCIATED WITH THE DPANN ARCHAEAL SUPERPHYLUM Article 24 October 2024 BACTERIAL C-DI-GMP HAS A KEY ROLE IN ESTABLISHING HOST–MICROBE SYMBIOSIS Article Open access 31 August

2023 DATA AVAILABILITY Metagenomic datasets generated from the CG20,27 ecosystem in 2009, 2014 and 2015 (_n_ = 66) and the HURL24 environment (_n_ = 2) were downloaded from the NCBI SRA in

April 2019 (Supplementary Table 1). SAGs generated in a previous study20 (_n_ = 219) were retrieved from the Joint Genome Institute’s Integrated Microbial Genomes and Microbiomes database111

(Supplementary Table 3). The metagenome-derived genomes of _Ca_. A. crystalense and _Ca_. H. crystalense from CG are publicly accessible from NCBI (accession numbers in Supplementary Table

2). The genomes of _Ca_. A. horonobense and _Ca_. H. julieae from HURL were newly reconstructed in this investigation (Supplementary Table 2). All previously unpublished genomes used in this

study are available in figshare https://doi.org/10.6084/m9.figshare.22339555(ref. 112) and all viral genomes are available at https://doi.org/10.6084/m9.figshare.22738568(ref. 113). All raw

FISH images are deposited here: https://doi.org/10.6084/m9.figshare.22739849(ref. 114). CODE AVAILABILITY The code used in this publication is based on previously published code. Please

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Kultur und Wissenschaft des Landes Nordrhein- Westfalen (Nachwuchsgruppe Dr. Alexander Probst) and the German Science Foundation under project NOVAC (grant no. DFG PR1603/2-1) and through

SPP 2141 (grant no. DFG BE6703/1-1). Genome-scale metabolic modelling was supported by the National Science Foundation under grant no. 1553211. The Ministry of Economy, Trade and Industry of

Japan funded a part of the work as ‘The project for validating assessment methodology in geological disposal system’ (2019 FY, grant no. JPJ007597). The work (proposal

https://doi.org/10.46936/10.25585/60000800) conducted by the US Department of Energy (DOE) Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is

supported by the Office of Science of the US DOE operated under contract no. DE-AC02-05CH11231. M.P. is supported by the Austrian Science Funds (project MAINTAIN, DOC 69 doc.funds). P.S.A.

was supported by a postdoctoral fellowship from the Alexander von Humboldt Foundation. J.P. was supported by Aker BP within the framework of the GeneOil Project given to A.J.P. J.R. was

supported by the German Science Foundation (grant no. RA3432/1-1, project no. 446702140). Support by the German Federal Ministry of Education and Research within the project ‘MultiKulti’

(BMBF funding code: 161L0285E) is acknowledged. We thank K. Dreger for exemplary server administration and B. Siebers, I. Berg, J. F. Banfield and B. Meyer for insightful discussions. AUTHOR

INFORMATION Author notes * Janina Rahlff Present address: Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Department of Biology and Environmental Science, Linnaeus

University, Kalmar, Sweden * Weishu Zhao Present address: Shanghai Jiao Tong University, School of Life Sciences and Biotechnology, International Center for Deep Life Investigation (IC-DLI),

Shanghai Jiao Tong University, Shanghai, China * Katrin Schwank Present address: University of Regensburg, Biochemistry III, Regensburg, Germany * These authors contributed equally: Sarah

P. Esser, Janina Rahlff. AUTHORS AND AFFILIATIONS * Environmental Metagenomics, Research Center One Health Ruhr of the University Alliance Ruhr, Faculty of Chemistry, University of

Duisburg-Essen, Essen, Germany Sarah P. Esser, Julia Plewka, Katharina Sures, Victoria Turzynski, Indra Banas, Till L. V. Bornemann, Perla Abigail Figueroa-Gonzalez & Alexander J. Probst

* Group for Aquatic Microbial Ecology, Environmental Microbiology and Biotechnology, University of Duisburg-Essen, Essen, Germany Sarah P. Esser, Janina Rahlff, Julia Plewka, Katharina

Sures, Panagiotis S. Adam, Victoria Turzynski, Indra Banas, Katrin Schwank, Till L. V. Bornemann, Perla Abigail Figueroa-Gonzalez & Alexander J. Probst * Department of Cell and Molecular

Biology, College of the Environment and Life Sciences, University of Rhode Island, Kingston, RI, USA Weishu Zhao & Ying Zhang * Computational Systems Biology, Centre for Microbiology

and Environmental Systems Science, University of Vienna, Vienna, Austria Michael Predl & Thomas Rattei * Doctoral School in Microbiology and Environmental Science, University of Vienna,

Vienna, Austria Michael Predl & Thomas Rattei * Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), Würzburg, Germany Franziska

Wimmer & Chase L. Beisel * DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Janey Lee, Jessica Jarett, Ian K. Blaby, Jan-Fang Cheng & Tanja Woyke

* School of Biological Sciences, University of Utah, Salt Lake City, UT, USA Julia McGonigle & William J. Brazelton * Institut Pasteur, Université Paris Cité, CNRS UMR6047, Archaeal

Virology Unit, Paris, France Mart Krupovic * Nuclear Fuel Cycle Engineering Laboratories, Japan Atomic Energy Agency, Tokai, Japan Yuki Amano * Medical faculty, University of Würzburg,

Würzburg, Germany Chase L. Beisel * Centre of Water and Environmental Research (ZWU), University of Duisburg-Essen, Essen, Germany Alexander J. Probst * Centre of Medical Biotechnology

(ZMB), University of Duisburg-Essen, Essen, Germany Alexander J. Probst Authors * Sarah P. Esser View author publications You can also search for this author inPubMed Google Scholar * Janina

Rahlff View author publications You can also search for this author inPubMed Google Scholar * Weishu Zhao View author publications You can also search for this author inPubMed Google

Scholar * Michael Predl View author publications You can also search for this author inPubMed Google Scholar * Julia Plewka View author publications You can also search for this author

inPubMed Google Scholar * Katharina Sures View author publications You can also search for this author inPubMed Google Scholar * Franziska Wimmer View author publications You can also search

for this author inPubMed Google Scholar * Janey Lee View author publications You can also search for this author inPubMed Google Scholar * Panagiotis S. Adam View author publications You

can also search for this author inPubMed Google Scholar * Julia McGonigle View author publications You can also search for this author inPubMed Google Scholar * Victoria Turzynski View

author publications You can also search for this author inPubMed Google Scholar * Indra Banas View author publications You can also search for this author inPubMed Google Scholar * Katrin

Schwank View author publications You can also search for this author inPubMed Google Scholar * Mart Krupovic View author publications You can also search for this author inPubMed Google

Scholar * Till L. V. Bornemann View author publications You can also search for this author inPubMed Google Scholar * Perla Abigail Figueroa-Gonzalez View author publications You can also

search for this author inPubMed Google Scholar * Jessica Jarett View author publications You can also search for this author inPubMed Google Scholar * Thomas Rattei View author publications

You can also search for this author inPubMed Google Scholar * Yuki Amano View author publications You can also search for this author inPubMed Google Scholar * Ian K. Blaby View author

publications You can also search for this author inPubMed Google Scholar * Jan-Fang Cheng View author publications You can also search for this author inPubMed Google Scholar * William J.

Brazelton View author publications You can also search for this author inPubMed Google Scholar * Chase L. Beisel View author publications You can also search for this author inPubMed Google

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Google Scholar * Alexander J. Probst View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.P.E. and A.J.P. performed genome-resolved

metagenomics, while S.P.E. and J.R. performed viromics. J.R. analysed viral genomes with input from M.K. CRISPR–Cas analyses were done by S.P.E., J.R. and A.J.P. SNP analysis was performed

by M.P. and T.R. Genome-scale modelling was conducted by W.Z. and Y.Z. with input from S.P.E., P.A.F.G. and A.J.P. J.P. conducted the sliding window analysis with the input from S.P.E.,

J.R., and A.J.P. Phylogenomic analyses were carried out by P.S.A. T.L.V.B. provided bioinformatic assistance and K. Schwank, I.B. and V.T. performed microscopy and initial metabolic

analyses. J.M. and W.B. resampled CG and J.L., T.W. and A.J.P. conducted RNA extraction and sequencing and S.P.E. analysed transcriptomes. J-F.C. synthesized the Cas genes with input from

I.K.B., F.W. and C.B. performed binding, cleavage and PAM assays and J.L., J.J., Y.A., T.W. and A.J.P. generated/provided raw data. K. Sures and S.P.E. analysed the archaeal CRISPR–Cas

interactions from published NCBI archaeal genomes. A.J.P. conceptualized the work. S.P.E., J.R., W.Z., Y.Z. and A.J.P. wrote the manuscript with input from all authors. CORRESPONDING AUTHOR

Correspondence to Alexander J. Probst. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Microbiology_ thanks

the anonymous reviewers for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims

in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 CORRELATION OF REPEAT ABUNDANCE AND ABUNDANCE OF CA. ALTIARCHAEA GENOMES. Spearman rank correlation

(two-tailed) of logarithmic abundances of _Ca_. A. crystalense and logarithmic abundances of repeat sequences of the unassigned CRISPR array (p-value < 3.4 e−16) and the CRISPR system

type I-B (p < 2.2 e−16) in metagenomes from CG (n = 66). The grey area depicts the confidence interval of 0.95. The line indicates that the correlation of the genome abundance and repeat

abundance is linear. Visualization was performed with R87,117 (version 3.6.1). EXTENDED DATA FIG. 2 VIRAL CLUSTERS PREDICTED BY VIRIDIC79. Heatmap showing intergenomic similarity for viral

scaffolds of viral clusters (VC_XY) and some singletons (black). Colouring of viral OTUs (vOTUs) according to Supplementary Table 6. VC_09, _12, _13 determined by the other tools were not

found by VIRIDIC. Only scaffolds with intergenomic similarity of >10 between two viral scaffolds are shown. EXTENDED DATA FIG. 3 COVERAGE ANALYSES OF SCAFFOLDS TARGETED BY SPACERS FROM

_CA_. ALTIARCHAEA. Coverage changes within targeted regions by CRISPR system type I-B of _Ca_. Altiarchaeum and _Ca_. Huberiarchaeum based on metagenomic read mapping. The vertically grey

marked regions are spacer targeted regions of either _Ca_. Altiarchaeum or _Ca_. Huberiarchaeum, whereby the horizontally dark grey lines are showing the average coverage of the scaffold.

The coloured graphs show the coverage across the spacer targeted region of three samples from the minor eruption phase, where _Ca_. Altiarchaeum is the most abundant organism (Supplementary

Fig. 1). EXTENDED DATA FIG. 4 SPACER TARGETING ANALYSES OF PUBLICLY AVAILABLE ARCHAEAL GENOMES. Directed spacer analysis of 7,012 publicly available archaeal genomes (Supplementary Table 4)

shows large clusters of spacers targeting at species level. The targeting spacers (edges) of the genomes _Sulfolobus_, _Methanomicrobia_ and _Halobacterium_ (nodes) form large clusters

performing self-targeting or targeting other genomes of the same family. Edges are colored according to their relationship at least familiy level or lower. The clustering was illustrated

with Cytoscape83 (version 3.9.1). Please note that targeting within the same genus might limit the interspecies recombination, as demonstrated in haloarchaea37, or reflect the presence of

multiple conserved genomic regions between the genomes. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Results, Table 7 and Figs. 1–9. REPORTING SUMMARY SUPPLEMENTARY DATA

Collection of all phylogenetic trees calculated for this study. (1) Phylogenetic tree of _Ca_. Altiarchaeum crystalense/horonobense and _Ca_. Huberiarchaeum crystalense/julieae located

within the archaeal branch. (2) Phylogenetic analysis of the phenylalanine-tRNA synthetase of _Ca_. Huberiarchaeum crystalense located within the archaeal branch. Calculated to determine the

closest relative within the archaeal branch. (3) Phylogenetic analysis of the phenylalanine-tRNA synthetase of _Ca_. Altiarchaeum crystalense located within the archaeal branch. Calculated

to determine the closest relative within the archaeal branch. (4) Phylogenetic analysis of the lysine-tRNA synthetase of _Ca_. Huberiarchaeum crystalense located within the archaeal branch.

Calculated to determine the closest relative within the archaeal branch. (5) Phylogenetic analysis of the lysine-tRNA synthetase of _Ca_. Altiarchaeum crystalense located within the archaeal

branch. Calculated to determine the closest relative within the archaeal branch. SUPPLEMENTARY TABLES Supplementary Tables 1–6 and 8–13. RIGHTS AND PERMISSIONS Springer Nature or its

licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the

accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Esser, S.P., Rahlff, J., Zhao, W. _et al._ A predicted CRISPR-mediated symbiosis between uncultivated archaea. _Nat Microbiol_ 8, 1619–1633 (2023). https://doi.org/10.1038/s41564-023-01439-2

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