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Title: New CRISPR–Cas systems from uncultivated microbes

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:
Grant/Contract Number:
AC02-05CH11231
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
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Protein function predictions; RNA; Microbiology; Microbial ecology; Biotechnology
OSTI Identifier:
1379729

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., 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.. 2016. "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}
}

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