CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
22 08 2019
22 08 2019
Historique:
accepted:
12
06
2019
revised:
07
06
2019
received:
09
05
2019
pubmed:
21
6
2019
medline:
18
12
2019
entrez:
21
6
2019
Statut:
ppublish
Résumé
Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.
Identifiants
pubmed: 31219587
pii: 5521030
doi: 10.1093/nar/gkz548
pmc: PMC6698737
doi:
Substances chimiques
Archaeal Proteins
0
CRISPR-Associated Proteins
0
Endonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
7518-7531Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM118140
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM118160
Pays : United States
Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
Références
Nucleic Acids Res. 2017 Nov 2;45(19):11281-11294
pubmed: 29036456
Curr Opin Microbiol. 2017 Jun;37:67-78
pubmed: 28605718
Nat Rev Microbiol. 2019 Jan;17(1):7-12
pubmed: 30171202
Science. 2008 Aug 15;321(5891):960-4
pubmed: 18703739
J Mol Evol. 2005 Feb;60(2):174-82
pubmed: 15791728
Nature. 2015 Mar 12;519(7542):193-8
pubmed: 25707795
RNA Biol. 2013 May;10(5):659-70
pubmed: 23422322
Extremophiles. 2017 Jan;21(1):95-107
pubmed: 27582008
Nucleic Acids Res. 2014 Jul;42(12):7884-93
pubmed: 24920831
Curr Opin Microbiol. 2017 Jun;37:110-119
pubmed: 28646675
Chembiochem. 2013 Feb 11;14(3):323-31
pubmed: 23355266
Genes Dev. 2016 Feb 15;30(4):447-59
pubmed: 26848045
J Bacteriol. 2008 Feb;190(4):1390-400
pubmed: 18065545
Nucleic Acids Res. 2015 Dec 2;43(21):10353-63
pubmed: 26519471
RNA Biol. 2013 May;10(5):792-802
pubmed: 23445770
Nature. 2015 Oct 1;526(7571):55-61
pubmed: 26432244
Genome Biol. 2009;10(3):R25
pubmed: 19261174
Elife. 2015 Aug 18;4:
pubmed: 26284603
BMC Bioinformatics. 2007 May 23;8:172
pubmed: 17521438
Nucleic Acids Res. 2015 Feb 18;43(3):1749-58
pubmed: 25589547
Q Rev Biophys. 2009 Feb;42(1):41-81
pubmed: 19508739
Nucleic Acids Res. 2017 Mar 17;45(5):2714-2723
pubmed: 28034956
Extremophiles. 2019 Jan;23(1):19-33
pubmed: 30284045
Mol Cell. 2016 Jun 16;62(6):824-833
pubmed: 27211867
Nucleic Acids Res. 2018 Feb 16;46(3):1007-1020
pubmed: 29228332
Cell. 2018 Mar 8;172(6):1239-1259
pubmed: 29522745
Nat Struct Mol Biol. 2014 Jun;21(6):528-34
pubmed: 24793649
Science. 2017 Apr 7;356(6333):
pubmed: 28385959
Nat Rev Microbiol. 2014 Jul;12(7):479-92
pubmed: 24909109
Science. 2007 Mar 23;315(5819):1709-12
pubmed: 17379808
RNA Biol. 2013 May;10(5):891-9
pubmed: 23403393
Genes Dev. 2014 Nov 1;28(21):2432-43
pubmed: 25367038
Mol Cell. 2018 Apr 5;70(1):48-59.e5
pubmed: 29602742
Nat Microbiol. 2018 Mar;3(3):310-318
pubmed: 29379209
Proc Natl Acad Sci U S A. 2017 Jun 27;114(26):E5122-E5128
pubmed: 28611213
Mol Cell. 2019 Feb 21;73(4):727-737.e3
pubmed: 30709710
Cell Rep. 2016 Sep 13;16(11):2811-2818
pubmed: 27626652
Genome Res. 2004 Jun;14(6):1188-90
pubmed: 15173120
Cell. 2015 Nov 5;163(4):840-53
pubmed: 26478180
Mol Cell. 2016 Mar 17;61(6):797-808
pubmed: 26949040
Nat Struct Mol Biol. 2016 Oct;23(10):876-883
pubmed: 27595346
Nat Rev Microbiol. 2015 Nov;13(11):722-36
pubmed: 26411297
Genes Dev. 2008 Dec 15;22(24):3489-96
pubmed: 19141480
Mol Cell. 2016 Nov 3;64(3):616-623
pubmed: 27618488
Mol Cell. 2018 Jun 7;70(5):814-824.e6
pubmed: 29883605
FEMS Microbiol Rev. 2015 May;39(3):428-41
pubmed: 25994611
Elife. 2019 Apr 30;8:
pubmed: 31021314
Cell. 2009 Nov 25;139(5):945-56
pubmed: 19945378
Nucleic Acids Res. 2012 Jul;40(12):5569-76
pubmed: 22402487
Science. 2017 Sep 15;357(6356):1113-1118
pubmed: 28729350
Bioinformatics. 2010 Mar 15;26(6):841-2
pubmed: 20110278
Nature. 2017 Oct 5;550(7674):137-141
pubmed: 28869593
J Bacteriol. 2012 May;194(10):2491-500
pubmed: 22408157
Nucleic Acids Res. 2017 Jan 9;45(1):367-381
pubmed: 27899566
Nucleic Acids Res. 2016 May 19;44(9):4266-77
pubmed: 27085805
Bioinformatics. 2016 Sep 1;32(17):i576-i585
pubmed: 27587677