A Programmable Dual-RNA-guided DNA Endonuclease in Adaptive Bacterial Immunity

Overview

Journal Science

Specialty Science

Date 2012 Jun 30

PMID 22745249

Citations 6848

Authors

Martin Jinek

Krzysztof Chylinski

Ines Fonfara

Michael Hauer

Jennifer A Doudna

Emmanuelle Charpentier

Affiliations

Soon will be listed here.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

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References

Hatoum-Aslan A, Maniv I, Marraffini L . Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci U S A. 2011; 108(52):21218-22. PMC: 3248500. DOI: 10.1073/pnas.1112832108. View

Deveau H, Barrangou R, Garneau J, Labonte J, Fremaux C, Boyaval P . Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2007; 190(4):1390-400. PMC: 2238228. DOI: 10.1128/JB.01412-07. View

Urnov F, Rebar E, Holmes M, Zhang H, Gregory P . Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010; 11(9):636-46. DOI: 10.1038/nrg2842. View

Gottesman S . Microbiology: Dicing defence in bacteria. Nature. 2011; 471(7340):588-9. DOI: 10.1038/471588a. View

Semenova E, Jore M, Datsenko K, Semenova A, Westra E, Wanner B . Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A. 2011; 108(25):10098-103. PMC: 3121866. DOI: 10.1073/pnas.1104144108. View

Sashital D, Wiedenheft B, Doudna J . Mechanism of foreign DNA selection in a bacterial adaptive immune system. Mol Cell. 2012; 46(5):606-15. PMC: 3397241. DOI: 10.1016/j.molcel.2012.03.020. View

Wiedenheft B, Lander G, Zhou K, Jore M, Brouns S, van der Oost J . Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature. 2011; 477(7365):486-489. PMC: 4165517. DOI: 10.1038/nature10402. View

Westra E, van Erp P, Kunne T, Wong S, Staals R, Seegers C . CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell. 2012; 46(5):595-605. PMC: 3372689. DOI: 10.1016/j.molcel.2012.03.018. View

Wiedenheft B, van Duijn E, Bultema J, Bultema J, Waghmare S, Waghmare S . RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc Natl Acad Sci U S A. 2011; 108(25):10092-7. PMC: 3121849. DOI: 10.1073/pnas.1102716108. View

10.

Garneau J, Dupuis M, Villion M, Romero D, Barrangou R, Boyaval P . The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010; 468(7320):67-71. DOI: 10.1038/nature09523. View

11.

Marraffini L, Sontheimer E . Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature. 2010; 463(7280):568-71. PMC: 2813891. DOI: 10.1038/nature08703. View

12.

Deltcheva E, Chylinski K, Sharma C, Gonzales K, Chao Y, Pirzada Z . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-7. PMC: 3070239. DOI: 10.1038/nature09886. View

13.

Hale C, Zhao P, Olson S, Duff M, Graveley B, Wells L . RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009; 139(5):945-56. PMC: 2951265. DOI: 10.1016/j.cell.2009.07.040. View

14.

Makarova K, Haft D, Barrangou R, Brouns S, Charpentier E, Horvath P . Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011; 9(6):467-77. PMC: 3380444. DOI: 10.1038/nrmicro2577. View

15.

Hutvagner G, Simard M . Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol. 2007; 9(1):22-32. DOI: 10.1038/nrm2321. View

16.

Terns M, Terns R . CRISPR-based adaptive immune systems. Curr Opin Microbiol. 2011; 14(3):321-7. PMC: 3119747. DOI: 10.1016/j.mib.2011.03.005. View

17.

Carroll D . Genome engineering with zinc-finger nucleases. Genetics. 2011; 188(4):773-82. PMC: 3176093. DOI: 10.1534/genetics.111.131433. View

18.

Lintner N, Kerou M, Brumfield S, Graham S, Liu H, Naismith J . Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). J Biol Chem. 2011; 286(24):21643-56. PMC: 3122221. DOI: 10.1074/jbc.M111.238485. View

19.

Wang R, Preamplume G, Terns M, Terns R, Li H . Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage. Structure. 2011; 19(2):257-64. PMC: 3154685. DOI: 10.1016/j.str.2010.11.014. View

20.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View