CRISPR–Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA1,2. Class 2 CRISPR–Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences3,4. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research5. However, current CRISPR–Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms6,7. Here, using genome-resolved metagenomics, we identify a number of CRISPR–Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR–Cas system. In bacteria, we discovered two previously unknown systems, CRISPR–CasX and CRISPR–CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.
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We thank N. Ma, K. Zhou and D. McGrath for technical assistance; C. Brown, M. Olm, M. O’Connell, J. Chen and S. Floor for reading the manuscript and discussions; and V. Yu for the S. cerevisiae expression strain. D.B. was supported by a long-term EMBO fellowship, L.B.H. by a US National Science Foundation Graduate Research Fellowship, and A.J.P. by a fellowship of the German Science Foundation (DFG PR 1603/1-1). J.A.D. is an Investigator of the Howard Hughes Medical Institute. This research was supported in part by the Allen Distinguished Investigator Program, through The Paul G. Allen Frontiers Group, the National Science Foundation (MCB-1244557 to J.A.D.) and the Lawrence Berkeley National Laboratory’s Sustainable Systems Scientific Focus Area funded by the US Department of Energy (DE-AC02-05CH11231 to J.F.B.). DNA sequencing was conducted at the DOE Joint Genome Institute, a DOE Office of Science User Facility, via the Community Science Program.
The Regents of the University of California have filed a provisional patent application related to the technology described in this work to the United States Patent and Trademark Office, in which D.B., L.B.H., S.C.S., J.A.D. and J.F.B. are listed as inventors.
Reviewer Information Nature thanks E. Sontheimer, R. Sorek and M. White for their contribution to the peer review of this work.
Extended data figures and tables
Alignment of Cas9 proteins from ARMAN-1 and ARMAN-4, as well as two closely related Cas9 proteins from uncultivated bacteria, to the Actinomyces naeslundii Cas9, whose structure has been solved67.
Variability of reconstructed CRISPR arrays, including the most well represented (and thus assembled) sequences (Fig. 2) and array segments representing locus variants that were reconstructed from the short DNA reads. Variability is due to spacers that were present in only a subset of archaeal cells in the population, as well as spacers whose context differed owing to spacer loss (indicated by black lines). White boxes indicate repeats and coloured arrows indicate CRISPR spacers (spacers with different colours have different sequences, except for unique spacers that are black). In CRISPR systems, spacers are typically added unidirectionally, so the high variety of spacers on the left side is attributed to recent acquisition.
a, Simplified phylogenetic tree of the universal Cas1 protein. CRISPR types of known systems are noted on the wedges and branches; the newly described systems are in bold. Detailed Cas1 phylogeny is provided in Supplementary Data 4. b, Proposed evolutionary scenario that gave rise to the archaeal type II system as a result of a recombination between type II-B and type II-C loci. c, Similarity of CasX and CasY to known proteins based on the following searches: (1) BLAST search against the non-redundant (NR) protein database of NCBI; (2) HMM search against an HMM database of known Cas proteins; and (3) distant homology search using HHpred49 (E, e value).
Maximum-likelihood phylogenic tree of Cas9 proteins, showing the previously described systems coloured based on their type. II-A, blue; II-B, green; II-C, purple. The archaeal Cas9 (red) cluster with type II-C CRISPR–Cas systems, together with two newly described bacterial Cas9 from uncultivated bacteria. A detailed tree is provided in Supplementary Data 5.
a, Protospacers from ARMAN-1 map to the genome of ARMAN-2, a nanoarchaeon from the same environment. Six protospacers (red arrowheads) map uniquely to a portion of the genome flanked by two long-terminal repeats (LTRs), and two additional protospacers match perfectly within the LTRs (blue and green arrowheads). This region is likely to be a transposon, suggesting that the CRISPR–Cas system of ARMAN-1 plays a role in suppressing mobilization of this element. b, Protospacers also map to a Thermoplasmatales archaeon (I-plasma), another member of the Richmond Mine ecosystem that is found in the same samples as ARMAN organisms. The protospacers cluster within a region of the genome encoding short, hypothetical proteins, suggesting this might also represent a mobile element. NCBI accession codes are provided in parentheses.
Extended Data Figure 6 Archaeal Cas9 from ARMAN-4 with a degenerate CRISPR array is found on numerous contigs.
Cas9 from ARMAN-4 is highlighted in dark red on 16 nearly identical contigs from different samples. Proteins with putative domains or functions are labelled, whereas hypothetical proteins are unlabelled. Fifteen of the contigs contain two degenerate direct repeats (36 nucleotides long with one mismatch) and a single conserved spacer of 36 nucleotides. The remaining contig contains only one direct repeat. Unlike ARMAN-1, no additional Cas proteins are found adjacent to Cas9 in ARMAN-4.
Extended Data Figure 7 Predicted structures of guide RNA and purification schema for in vitro biochemistry studies.
a, The CRISPR repeat and tracrRNA anti-repeat are depicted in black whereas the spacer-derived sequence is shown as a series of green Ns. No clear termination signal can be predicted from the locus, so three different tracrRNA lengths were tested based on their secondary structure: 69, 104, and 179 nucleotides in red, blue, and pink, respectively. b, Engineered single-guide RNA corresponding to dual-guide in a. c, Dual-guide RNA for ARMAN-4 Cas9 with two different hairpins on 3′ end of tracrRNA (75 and 122 nucleotides). d, Engineered single-guide RNA corresponding to dual-guide in c. e, Conditions tested in E. coli in vivo targeting assay. f, ARMAN-1 (AR1) and ARMAN-4 (AR4) Cas9 were expressed and purified under a variety of conditions as outlined in the Methods section. Proteins outlined in blue boxes were tested for cleavage activity in vitro. g, Fractions of AR1-Cas9 and AR4-Cas9 purifications were separated on a 10% SDS–PAGE gel.
a, Plasmid interference assays for CasX.1 (Deltaproteobacteria) and CasX.2 (Planctomycetes), continued from Fig. 3c (sX1, CasX spacer 1; sX2, CasX spacer 2; NT, non-target). Experiments were conducted in triplicate and mean ± s.d. is shown. b, Serial dilution of E. coli expressing a CasX locus and transformed with the specified target, continued from Fig. 3b. c, PAM depletion assays for the Deltaproteobacteria CasX and d, Planctomycetes CasX expressed in E. coli. PAM sequences depleted greater than the indicated PAM depletion value threshold (PDVT) compared to a control library were used to generate the sequence logo. e, Diagram depicting the location of northern blot probes for CasX.1. f, Northern blots for CasX.1 tracrRNA in total RNA extracted from E. coli expressing the CasX.1 locus. The sequences of the probes used are provided in Supplementary Table 2.
This file contains Supplementary Table 1, reconstructed spacer and protospacers of the ARMAN-1 Type II CRISPR-Cas system. (XLSX 31 kb)
This file contains Supplementary Table 2, a list of primers and plasmids used in the study. (XLSX 38 kb)
This zipped file contains Supplementary Data sets 1-6. (ZIP 10208 kb)
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Burstein, D., Harrington, L., Strutt, S. et al. New CRISPR–Cas systems from uncultivated microbes. Nature 542, 237–241 (2017). https://doi.org/10.1038/nature21059
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