title: Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.
abstract: In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.
authors: Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
authors: Reuben B
authors: RB
authors: Vercoe
authors: James T
authors: JT
authors: Chang
authors: Ron L
authors: RL
authors: Dy
authors: Corinda
authors: C
authors: Taylor
authors: Tamzin
authors: T
authors: Gristwood
authors: James S
authors: JS
authors: Clulow
authors: Corinna
authors: C
authors: Richter
authors: Rita
authors: R
authors: Przybilski
authors: Andrew R
authors: AR
authors: Pitman
authors: Peter C
authors: PC
authors: Fineran
mesh_terms: D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D016680:Genome, Bacterial; D044404:Genomic Islands; D010957:Plasmids
references: Int J Food Microbiol. 2009 Apr 30;131(1):62-70
references: 18635282
references: Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):894-9
references: 19124776
references: Curr Opin Microbiol. 2003 Oct;6(5):506-11
references: 14572544
references: Annu Rev Genet. 2012;46:311-39
references: 23145983
references: J Bacteriol. 2004 Dec;186(23):8114-22
references: 15547285
references: Science. 2010 Jan 8;327(5962):167-70
references: 20056882
references: Environ Microbiol. 2008 May;10(5):1202-17
references: 18294208
references: Curr Biol. 2009 Sep 29;19(18):1586-90
references: 19747826
references: Nat Commun. 2012 Jul 10;3:945
references: 22781758
references: J Bacteriol. 2010 Dec;192(23):6291-4
references: 20889749
references: PLoS One. 2012;7(4):e35888
references: 22558257
references: J Biol Chem. 2011 Jun 17;286(24):21643-56
references: 21507944
references: Mol Cell. 2012 Feb 10;45(3):303-13
references: 22227115
references: Mol Microbiol. 2002 Mar;43(6):1565-75
references: 11952905
references: EMBO J. 2011 Oct 18;30(22):4616-27
references: 22009198
references: Nature. 1981 Apr 30;290(5809):797-9
references: 7012641
references: J Bacteriol. 2008 Feb;190(4):1390-400
references: 18065545
references: Mol Cell. 2012 Feb 10;45(3):292-302
references: 22227116
references: J Biol Chem. 2011 Sep 9;286(36):31896-903
references: 21775431
references: J Bacteriol. 2012 Nov;194(21):5728-38
references: 22885297
references: Microbiology (Reading). 2009 Mar;155(Pt 3):733-740
references: 19246744
references: RNA. 2012 Apr;18(4):661-72
references: 22345129
references: PLoS One. 2012;7(12):e49549
references: 23226499
references: Nat Biotechnol. 2013 Mar;31(3):233-9
references: 23360965
references: BMC Bioinformatics. 2007 May 23;8:172
references: 17521438
references: Microbiology (Reading). 2010 May;156(5):1351-1361
references: 28206910
references: Science. 2010 Sep 10;329(5997):1355-8
references: 20829488
references: Nat Rev Genet. 2010 Mar;11(3):181-90
references: 20125085
references: Structure. 2012 Sep 5;20(9):1574-84
references: 22841292
references: Mol Gen Genet. 1988 Aug;213(2-3):491-8
references: 2972909
references: FEMS Microbiol Rev. 2004 May;28(2):127-81
references: 15109783
references: Mol Microbiol. 2012 Sep;85(6):1057-71
references: 22834929
references: Science. 2008 Dec 19;322(5909):1843-5
references: 19095942
references: Plasmid. 2002 Nov;48(3):202-12
references: 12460536
references: Nat Rev Microbiol. 2004 May;2(5):414-24
references: 15100694
references: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10098-103
references: 21646539
references: Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20136-41
references: 22114197
references: Nat Rev Microbiol. 2008 Mar;6(3):181-6
references: 18157154
references: Biol Direct. 2011 Dec 21;6(1):65
references: 22188759
references: Science. 2007 Mar 23;315(5819):1709-12
references: 17379808
references: J Bacteriol. 2010 Jul;192(13):3249-58
references: 20363934
references: EMBO J. 2011 Apr 6;30(7):1335-42
references: 21343909
references: Curr Biol. 2005 Dec 20;15(24):2230-5
references: 16360685
references: Viruses. 2012 Oct 19;4(10):2291-311
references: 23202464
references: Trends Biotechnol. 2007 Jan;25(1):7-15
references: 17113664
references: Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2141-6
references: 20133858
references: PLoS One. 2012;7(11):e50797
references: 23189210
references: RNA Biol. 2011 May-Jun;8(3):517-28
references: 21519197
references: J Bacteriol. 2011 Jul;193(14):3433-45
references: 21398535
references: Mol Cell. 2012 Jun 8;46(5):595-605
references: 22521689
references: RNA. 2008 Dec;14(12):2572-9
references: 18971321
references: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10092-7
references: 21536913
references: Microbiology (Reading). 2005 Dec;151(Pt 12):3833-3845
references: 16339930
references: Biol Direct. 2006 Mar 16;1:7
references: 16545108
references: Genome Biol. 2007;8(4):R61
references: 17442114
references: J Bacteriol. 2011 May;193(10):2460-7
references: 21421763
references: Science. 2008 Aug 15;321(5891):960-4
references: 18703739
references: Archaea. 2006 Aug;2(1):59-72
references: 16877322
references: Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):11105-10
references: 15263089
references: Biol Direct. 2011 Jul 14;6:38
references: 21756346
references: PLoS Comput Biol. 2005 Nov;1(6):e60
references: 16292354
references: Mol Microbiol. 2011 Apr;80(2):481-91
references: 21385233
references: Nature. 2010 Nov 4;468(7320):67-71
references: 21048762
references: Infect Immun. 2006 Sep;74(9):5272-83
references: 16926421
references: Virology. 2012 Dec 20;434(2):202-9
references: 23123013
references: Genes Dev. 2008 Dec 15;22(24):3489-96
references: 19141480
references: BMC Evol Biol. 2010 Jul 28;10:230
references: 20667132
references: PLoS One. 2010 Jun 15;5(6):e11126
references: 20559554
references: Nature. 2011 Mar 31;471(7340):602-7
references: 21455174
references: Cell. 2009 Nov 25;139(5):945-56
references: 19945378
references: Nat Rev Microbiol. 2010 May;8(5):317-27
references: 20348932
references: Nucleic Acids Res. 2012 Jul;40(12):5569-76
references: 22402487
references: Nat Rev Microbiol. 2011 Jun;9(6):467-77
references: 21552286
references: PLoS Pathog. 2008 Jun 20;4(6):e1000093
references: 18566662
references: Gene. 1991 Dec 20;109(1):137-41
references: 1756974
references: J Infect Dev Ctries. 2008 Aug 30;2(4):267-71
references: 19741287
references: Mol Microbiol. 2012 May;84(4):648-63
references: 22524709
references: Trends Genet. 2010 Aug;26(8):335-40
references: 20598393