title: New CRISPR-Cas systems from uncultivated microbes.
abstract: 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. 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.
authors.affiliation: Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.
authors.forename: David
authors.initials: D
authors.lastname: Burstein
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Harrington
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Strutt
authors.affiliation: Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Probst
authors.affiliation: Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.
authors.forename: Karthik
authors.initials: K
authors.lastname: Anantharaman
authors.affiliation: Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Thomas
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.|Department of Chemistry, University of California, Berkeley, California 94720, USA.|Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.|Innovative Genomics Initiative, University of California, Berkeley, California 94720, USA.|MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.|Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720, USA.
authors.forename: Jillian F
authors.initials: JF
authors.lastname: Banfield
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics*; D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D001709:Biotechnology / Q000639:trends; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics* / Q000502:physiology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D016678:Genome / Q000235:genetics; D056186:Metagenomics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D064130:CRISPR-Associated Proteins; D012329:RNA, Bacterial; D004247:DNA