title: Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis.
abstract: CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5' end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of Georgia, Athens, GA, USA.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Elmore
authors.forename: Yuusuke
authors.initials: Y
authors.lastname: Yokooji
authors.forename: Takaaki
authors.initials: T
authors.lastname: Sato
authors.forename: Sara
authors.initials: S
authors.lastname: Olson
authors.forename: Claiborne V C
authors.initials: CV
authors.lastname: Glover
authors.forename: Brenton R
authors.initials: BR
authors.lastname: Graveley
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Atomi
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Terns
authors.forename: Michael P
authors.initials: MP
authors.lastname: Terns
mesh_terms: D023641:Archaeal Viruses / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019848:Gene Expression Regulation, Archaeal; D005818:Genetic Engineering; D059014:High-Throughput Nucleotide Sequencing; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D019642:RNA, Archaeal / Q000235:genetics*; D017423:Sequence Analysis, RNA; D019712:Thermococcus / Q000235:genetics* / Q000378:metabolism
chemical_list: D019642:RNA, Archaeal
keywords: CRISPR; Cas; DNA; RNA; Thermococcus; archaea; hyperthermophile; immune; interference; silencing
references.citation: Nat Struct Mol Biol. 2011 May;18(5):529-36
references.pmid: 21460843
references.citation: Nature. 2011 Mar 31;471(7340):602-7
references.pmid: 21455174
references.citation: J Bacteriol. 2012 May;194(10):2491-500
references.pmid: 22408157
references.citation: J Bacteriol. 2003 Jan;185(1):210-20
references.pmid: 12486058
references.citation: J Biol Chem. 2001 Sep 21;276(38):35629-35
references.pmid: 11468293
references.citation: Curr Opin Microbiol. 2011 Jun;14(3):321-7
references.pmid: 21531607
references.citation: RNA. 2012 Nov;18(11):2020-8
references.pmid: 23006625
references.citation: PLoS One. 2012;7(4):e35888
references.pmid: 22558257
references.citation: Annu Rev Microbiol. 2010;64:475-93
references.pmid: 20528693
references.citation: J Biol Chem. 2011 Jun 17;286(24):21643-56
references.pmid: 21507944
references.citation: Mol Cell. 2012 Feb 10;45(3):303-13
references.pmid: 22227115
references.citation: Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21218-22
references.pmid: 22160698
references.citation: Mol Microbiol. 2002 Mar;43(6):1565-75
references.pmid: 11952905
references.citation: EMBO J. 2011 Oct 18;30(22):4616-27
references.pmid: 22009198
references.citation: J Biotechnol. 2006 Nov 1;126(2):186-95
references.pmid: 16730835
references.citation: J Bacteriol. 2008 Feb;190(4):1390-400
references.pmid: 18065545
references.citation: Mol Cell. 2012 Feb 10;45(3):292-302
references.pmid: 22227116
references.citation: J Biol Chem. 2011 Sep 9;286(36):31896-903
references.pmid: 21775431
references.citation: J Bacteriol. 2012 Nov;194(21):5728-38
references.pmid: 22885297
references.citation: RNA Biol. 2012 Sep;9(9):1134-8
references.pmid: 22954644
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Bioinformatics. 2009 Dec 15;25(24):3317-8
references.pmid: 19846435
references.citation: Structure. 2011 Feb 9;19(2):257-64
references.pmid: 21300293
references.citation: Microbiology (Reading). 2009 Mar;155(Pt 3):733-740
references.pmid: 19246744
references.citation: BMC Bioinformatics. 2007 May 23;8:172
references.pmid: 17521438
references.citation: Science. 2010 Sep 10;329(5997):1355-8
references.pmid: 20829488
references.citation: Annu Rev Food Sci Technol. 2012;3:143-62
references.pmid: 22224556
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86
references.pmid: 22949671
references.citation: Nature. 2012 Feb 15;482(7385):331-8
references.pmid: 22337052
references.citation: Nat Struct Mol Biol. 2011 Jun;18(6):688-92
references.pmid: 21572444
references.citation: Annu Rev Genet. 2012;46:311-39
references.pmid: 23145983
references.citation: FEMS Microbiol Lett. 2009 Jul;296(1):110-6
references.pmid: 19459963
references.citation: Nat Rev Genet. 2010 Mar;11(3):181-90
references.pmid: 20125085
references.citation: Structure. 2012 Sep 5;20(9):1574-84
references.pmid: 22841292
references.citation: Science. 2008 Dec 19;322(5909):1843-5
references.pmid: 19095942
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10098-103
references.pmid: 21646539
references.citation: J Biol Chem. 2012 Jun 29;287(27):22445-9
references.pmid: 22621933
references.citation: Mol Microbiol. 2010 Sep;77(6):1367-79
references.pmid: 20624226
references.citation: Science. 2007 Mar 23;315(5819):1709-12
references.pmid: 17379808
references.citation: Appl Environ Microbiol. 2008 May;74(10):3099-104
references.pmid: 18378640
references.citation: Nucleic Acids Res. 2012 Oct;40(19):9887-96
references.pmid: 22879377
references.citation: Mol Cell. 2012 Jun 8;46(5):606-15
references.pmid: 22521690
references.citation: Mol Microbiol. 2010 Sep;77(6):1380-93
references.pmid: 20659289
references.citation: PLoS One. 2012;7(4):e35306
references.pmid: 22514725
references.citation: J Biol Chem. 2009 Oct 9;284(41):28137-28145
references.pmid: 19666462
references.citation: Appl Environ Microbiol. 2011 Apr;77(7):2392-8
references.pmid: 21278271
references.citation: EMBO J. 2011 Apr 6;30(7):1335-42
references.pmid: 21343909
references.citation: Front Microbiol. 2012 Jul 13;3:251
references.pmid: 22811677
references.citation: Nature. 2011 Sep 21;477(7365):486-489
references.pmid: 21938068
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D646-52
references.pmid: 22080555
references.citation: Mol Microbiol. 2011 Jan;79(1):35-49
references.pmid: 21166892
references.citation: Mol Microbiol. 2010 Mar;75(6):1495-512
references.pmid: 20132443
references.citation: Mol Cell. 2012 Jun 8;46(5):595-605
references.pmid: 22521689
references.citation: PLoS One. 2012;7(11):e50797
references.pmid: 23189210
references.citation: RNA Biol. 2011 May-Jun;8(3):517-28
references.pmid: 21519197
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10092-7
references.pmid: 21536913
references.citation: Appl Environ Microbiol. 2005 Jul;71(7):3889-99
references.pmid: 16000802
references.citation: RNA. 2008 Dec;14(12):2572-9
references.pmid: 18971321
references.citation: PLoS Comput Biol. 2012;8(4):e1002475
references.pmid: 22532794
references.citation: Annu Rev Genet. 2011;45:273-97
references.pmid: 22060043
references.citation: Science. 2008 Aug 15;321(5891):960-4
references.pmid: 18703739
references.citation: J Bacteriol. 2004 Sep;186(17):5799-807
references.pmid: 15317785
references.citation: Genome Biol. 2007;8(4):R61
references.pmid: 17442114
references.citation: PLoS Comput Biol. 2005 Nov;1(6):e60
references.pmid: 16292354
references.citation: Mol Microbiol. 2011 Apr;80(2):481-91
references.pmid: 21385233
references.citation: Nature. 2010 Nov 4;468(7320):67-71
references.pmid: 21048762
references.citation: J Mol Biol. 2011 Jan 28;405(4):939-55
references.pmid: 21093452
references.citation: Science. 2012 Aug 17;337(6096):816-21
references.pmid: 22745249
references.citation: Trends Microbiol. 2011 Nov;19(11):549-56
references.pmid: 21945420
references.citation: Virology. 2012 Dec 20;434(2):202-9
references.pmid: 23123013
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13668-73
references.pmid: 10570130
references.citation: J Biol Chem. 2012 Sep 28;287(40):33351-63
references.pmid: 22767603
references.citation: Genes Dev. 2008 Dec 15;22(24):3489-96
references.pmid: 19141480
references.citation: Nat Commun. 2012 Jul 10;3:945
references.pmid: 22781758
references.citation: Nature. 2010 Jan 28;463(7280):568-71
references.pmid: 20072129
references.citation: Appl Environ Microbiol. 1994 Dec;60(12):4559-66
references.pmid: 7811092
references.citation: Cell. 2009 Nov 25;139(5):945-56
references.pmid: 19945378
references.citation: RNA. 2010 Nov;16(11):2181-8
references.pmid: 20884784
references.citation: Nucleic Acids Res. 2012 Jul;40(12):5569-76
references.pmid: 22402487
references.citation: Nat Rev Microbiol. 2011 Jun;9(6):467-77
references.pmid: 21552286
references.citation: Genome Biol. 2012 Jul 18;13(7):R63
references.pmid: 22809431
references.citation: Mol Microbiol. 2012 Sep;85(6):1044-56
references.pmid: 22834906
references.citation: Mol Microbiol. 2009 Apr;72(1):259-72
references.pmid: 19239620