skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: New CRISPR–Cas systems from uncultivated microbes

Abstract

We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. 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 organisms. 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 inmore » Escherichia coli. Lastly, 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.« less

Authors:
 [1];  [2];  [2];  [1];  [1];  [1];  [3];  [4]
  1. Univ. of California, Berkeley, CA (United States). Department of Earth and Planetary Sciences
  2. Univ. of California, Berkeley, CA (United States). Department of Molecular and Cell Biology
  3. Univ. of California, Berkeley, CA (United States). Department of Molecular and Cell Biology, Howard Hughes Medical Institute, Innovative Genomics Initiative, and MBIB Division
  4. Univ. of California, Berkeley, CA (United States). Department of Earth and Planetary Sciences and Department of Environmental Science, Policy, and Management
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1379729
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Nature (London)
Additional Journal Information:
Journal Name: Nature (London); Journal Volume: 542; Journal Issue: 7640; Journal ID: ISSN 0028-0836
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Protein function predictions; RNA; Microbiology; Microbial ecology; Biotechnology

Citation Formats

Burstein, David, Harrington, Lucas B., Strutt, Steven C., Probst, Alexander J., Anantharaman, Karthik, Thomas, Brian C., Doudna, Jennifer A., and Banfield, Jillian F. New CRISPR–Cas systems from uncultivated microbes. United States: N. p., 2016. Web. doi:10.1038/nature21059.
Burstein, David, Harrington, Lucas B., Strutt, Steven C., Probst, Alexander J., Anantharaman, Karthik, Thomas, Brian C., Doudna, Jennifer A., & Banfield, Jillian F. New CRISPR–Cas systems from uncultivated microbes. United States. doi:10.1038/nature21059.
Burstein, David, Harrington, Lucas B., Strutt, Steven C., Probst, Alexander J., Anantharaman, Karthik, Thomas, Brian C., Doudna, Jennifer A., and Banfield, Jillian F. Thu . "New CRISPR–Cas systems from uncultivated microbes". United States. doi:10.1038/nature21059. https://www.osti.gov/servlets/purl/1379729.
@article{osti_1379729,
title = {New CRISPR–Cas systems from uncultivated microbes},
author = {Burstein, David and Harrington, Lucas B. and Strutt, Steven C. and Probst, Alexander J. and Anantharaman, Karthik and Thomas, Brian C. and Doudna, Jennifer A. and Banfield, Jillian F.},
abstractNote = {We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. 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 organisms. 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. Lastly, 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.},
doi = {10.1038/nature21059},
journal = {Nature (London)},
number = 7640,
volume = 542,
place = {United States},
year = {2016},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 103 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis
journal, May 2013


An updated evolutionary classification of CRISPR–Cas systems
journal, September 2015

  • Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S.
  • Nature Reviews Microbiology, Vol. 13, Issue 11, p. 722-736
  • DOI: 10.1038/nrmicro3569

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation
journal, February 2014


Lineages of Acidophilic Archaea Revealed by Community Genomic Analysis
journal, December 2006


DNase H Activity of Neisseria meningitidis Cas9
journal, October 2015


Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems
journal, February 2016

  • Burstein, David; Sun, Christine L.; Brown, Christopher T.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms10613

CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats
journal, May 2007

  • Grissa, I.; Vergnaud, G.; Pourcel, C.
  • Nucleic Acids Research, Vol. 35, Issue Web Server
  • DOI: 10.1093/nar/gkm360

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch
journal, May 2016

  • Oakes, Benjamin L.; Nadler, Dana C.; Flamholz, Avi
  • Nature Biotechnology, Vol. 34, Issue 6
  • DOI: 10.1038/nbt.3528

Community-wide analysis of microbial genome sequence signatures
journal, January 2009

  • Dick, Gregory J.; Andersson, Anders F.; Baker, Brett J.
  • Genome Biology, Vol. 10, Issue 8
  • DOI: 10.1186/gb-2009-10-8-r85

The Phyre2 web portal for protein modeling, prediction and analysis
journal, May 2015

  • Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M.
  • Nature Protocols, Vol. 10, Issue 6
  • DOI: 10.1038/nprot.2015.053

Mfold web server for nucleic acid folding and hybridization prediction
journal, July 2003


Protospacer recognition motifs: Mixed identities and functional diversity
journal, February 2013

  • Shah, Shiraz A.; Erdmann, Susanne; Mojica, Francisco J. M.
  • RNA Biology, Vol. 10, Issue 5
  • DOI: 10.4161/rna.23764

EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data
journal, May 2011

  • Miller, Christopher S.; Baker, Brett J.; Thomas, Brian C.
  • Genome Biology, Vol. 12, Issue 5
  • DOI: 10.1186/gb-2011-12-5-r44

Classification and evolution of type II CRISPR-Cas systems
journal, April 2014

  • Chylinski, Krzysztof; Makarova, Kira S.; Charpentier, Emmanuelle
  • Nucleic Acids Research, Vol. 42, Issue 10
  • DOI: 10.1093/nar/gku241

CD-HIT: accelerated for clustering the next-generation sequencing data
journal, October 2012


CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes
journal, March 2007


JPred4: a protein secondary structure prediction server
journal, April 2015

  • Drozdetskiy, Alexey; Cole, Christian; Procter, James
  • Nucleic Acids Research, Vol. 43, Issue W1
  • DOI: 10.1093/nar/gkv332

CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea
journal, March 2008

  • Sorek, Rotem; Kunin, Victor; Hugenholtz, Philip
  • Nature Reviews Microbiology, Vol. 6, Issue 3
  • DOI: 10.1038/nrmicro1793

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease
journal, July 2014

  • Anders, Carolin; Niewoehner, Ole; Duerst, Alessia
  • Nature, Vol. 513, Issue 7519
  • DOI: 10.1038/nature13579

Prodigal: prokaryotic gene recognition and translation initiation site identification
journal, March 2010


CRISPR adaptation biases explain preference for acquisition of foreign DNA
journal, April 2015

  • Levy, Asaf; Goren, Moran G.; Yosef, Ido
  • Nature, Vol. 520, Issue 7548
  • DOI: 10.1038/nature14302

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector
journal, June 2016

  • Abudayyeh, Omar O.; Gootenberg, Jonathan S.; Konermann, Silvana
  • Science, Vol. 353, Issue 6299
  • DOI: 10.1126/science.aaf5573

MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets
journal, October 2015


Self-targeting by CRISPR: gene regulation or autoimmunity?
journal, August 2010


Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems
journal, November 2015


Applications of CRISPR technologies in research and beyond
journal, September 2016

  • Barrangou, Rodolphe; Doudna, Jennifer A.
  • Nature Biotechnology, Vol. 34, Issue 9
  • DOI: 10.1038/nbt.3659

Orthogonal Cas9 proteins for RNA-guided gene regulation and editing
journal, September 2013

  • Esvelt, Kevin M.; Mali, Prashant; Braff, Jonathan L.
  • Nature Methods, Vol. 10, Issue 11
  • DOI: 10.1038/nmeth.2681

MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability
journal, January 2013

  • Katoh, K.; Standley, D. M.
  • Molecular Biology and Evolution, Vol. 30, Issue 4
  • DOI: 10.1093/molbev/mst010

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

WebLogo: A Sequence Logo Generator
journal, May 2004

  • Crooks, Gavin E.; Hon, Gary; Chandonia, John-Marc
  • Genome Research, Vol. 14, Issue 6, p. 1188-1190
  • DOI: 10.1101/gr.849004

CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
journal, March 2011

  • Deltcheva, Elitza; Chylinski, Krzysztof; Sharma, Cynthia M.
  • Nature, Vol. 471, Issue 7340
  • DOI: 10.1038/nature09886

Small Genomes and Sparse Metabolisms of Sediment-Associated Bacteria from Four Candidate Phyla
journal, October 2013


Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System
journal, October 2015

  • Zetsche, Bernd; Gootenberg, Jonathan S.; Abudayyeh, Omar O.
  • Cell, Vol. 163, Issue 3, p. 759-771
  • DOI: 10.1016/j.cell.2015.09.038

Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees
journal, April 2016

  • Letunic, Ivica; Bork, Peer
  • Nucleic Acids Research, Vol. 44, Issue W1
  • DOI: 10.1093/nar/gkw290

RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies
journal, January 2014


IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth
journal, April 2012


Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea
journal, January 2013

  • Yelton, Alexis P.; Comolli, Luis R.; Justice, Nicholas B.
  • BMC Genomics, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2164-14-485

Metagenomic analysis of a high carbon dioxide subsurface microbial community populated by chemolithoautotrophs and bacteria and archaea from candidate phyla: High CO
journal, April 2015

  • Emerson, Joanne B.; Thomas, Brian C.; Alvarez, Walter
  • Environmental Microbiology, Vol. 18, Issue 6
  • DOI: 10.1111/1462-2920.12817

Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity
journal, May 2014

  • Nuñez, James K.; Kranzusch, Philip J.; Noeske, Jonas
  • Nature Structural & Molecular Biology, Vol. 21, Issue 6
  • DOI: 10.1038/nsmb.2820

Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity
journal, February 2015

  • Nuñez, James K.; Lee, Amy S. Y.; Engelman, Alan
  • Nature, Vol. 519, Issue 7542
  • DOI: 10.1038/nature14237

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Unusual biology across a group comprising more than 15% of domain Bacteria
journal, June 2015

  • Brown, Christopher T.; Hug, Laura A.; Thomas, Brian C.
  • Nature, Vol. 523, Issue 7559
  • DOI: 10.1038/nature14486

The crystal structure of Cpf1 in complex with CRISPR RNA
journal, April 2016


The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer Among Prokaryotes
journal, April 2006


Fast gapped-read alignment with Bowtie 2
journal, March 2012

  • Langmead, Ben; Salzberg, Steven L.
  • Nature Methods, Vol. 9, Issue 4
  • DOI: 10.1038/nmeth.1923

Genomes from Metagenomics
journal, November 2013


A new view of the tree of life
journal, April 2016


BLAST+: architecture and applications
journal, January 2009

  • Camacho, Christiam; Coulouris, George; Avagyan, Vahram
  • BMC Bioinformatics, Vol. 10, Issue 1
  • DOI: 10.1186/1471-2105-10-421

Mechanism of substrate selection by a highly specific CRISPR endoribonuclease
journal, February 2012


Interaction between Bacteriophage DMS3 and Host CRISPR Region Inhibits Group Behaviors of Pseudomonas aeruginosa
journal, October 2008

  • Zegans, M. E.; Wagner, J. C.; Cady, K. C.
  • Journal of Bacteriology, Vol. 191, Issue 1
  • DOI: 10.1128/JB.00797-08

Diverse uncultivated ultra-small bacterial cells in groundwater
journal, February 2015

  • Luef, Birgit; Frischkorn, Kyle R.; Wrighton, Kelly C.
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms7372

In Situ Evolutionary Rate Measurements Show Ecological Success of Recently Emerged Bacterial Hybrids
journal, April 2012


HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment
journal, December 2011

  • Remmert, Michael; Biegert, Andreas; Hauser, Andreas
  • Nature Methods, Vol. 9, Issue 2
  • DOI: 10.1038/nmeth.1818

Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA
journal, May 2016


A Distinct Small RNA Pathway Silences Selfish Genetic Elements in the Germline
journal, July 2006


Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli
journal, February 2012

  • Yosef, Ido; Goren, Moran G.; Qimron, Udi
  • Nucleic Acids Research, Vol. 40, Issue 12
  • DOI: 10.1093/nar/gks216

An efficient algorithm for large-scale detection of protein families
journal, April 2002


HMMER web server: interactive sequence similarity searching
journal, May 2011

  • Finn, R. D.; Clements, J.; Eddy, S. R.
  • Nucleic Acids Research, Vol. 39, Issue suppl
  • DOI: 10.1093/nar/gkr367

Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system
journal, October 2016

  • Anantharaman, Karthik; Brown, Christopher T.; Hug, Laura A.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms13219

Insights into the phylogeny and coding potential of microbial dark matter
journal, July 2013

  • Rinke, Christian; Schwientek, Patrick; Sczyrba, Alexander
  • Nature, Vol. 499, Issue 7459
  • DOI: 10.1038/nature12352

Crass: identification and reconstruction of CRISPR from unassembled metagenomic data
journal, March 2013

  • Skennerton, Connor T.; Imelfort, Michael; Tyson, Gene W.
  • Nucleic Acids Research, Vol. 41, Issue 10
  • DOI: 10.1093/nar/gkt183

The reduced genomes of Parcubacteria (OD1) contain signatures of a symbiotic lifestyle
journal, July 2015


Enigmatic, ultrasmall, uncultivated Archaea
journal, April 2010

  • Baker, B. J.; Comolli, L. R.; Dick, G. J.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 19
  • DOI: 10.1073/pnas.0914470107

    Works referencing / citing this record:

    Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems
    journal, June 2018


    Prediction of activity and specificity of CRISPR-Cpf1 using convolutional deep learning neural networks
    journal, June 2019


    Prediction and diversity of tracrRNAs from type II CRISPR-Cas systems
    journal, June 2018


    The emerging and uncultivated potential of CRISPR technology in plant science
    journal, July 2019


    The emerging and uncultivated potential of CRISPR technology in plant science
    journal, July 2019


    Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems
    journal, June 2018


    Prediction and diversity of tracrRNAs from type II CRISPR-Cas systems
    journal, June 2018


    Prediction of activity and specificity of CRISPR-Cpf1 using convolutional deep learning neural networks
    journal, June 2019


    Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease
    journal, January 2020