A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Martin Jinek,Krzysztof Chylinski,Krzysztof Chylinski,Ines Fonfara,Michael H. Hauer,Jennifer A. Doudna,Emmanuelle Charpentier +6 more
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TLDR
This 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.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.read more
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Journal ArticleDOI
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,Le Cong,F. Ann Ran,F. Ann Ran,David M. Cox,David M. Cox,Shuailiang Lin,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +15 more
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Multiplex Genome Engineering Using CRISPR/Cas Systems
Le Cong,F. A. Ran,David Benjamin Turitz Cox,Shuailiang Lin,Robert P. J. Barretto,Naomi Habib,Patrick D. Hsu,Xuebing Wu,Wenyan Jiang,Luciano A. Marraffini,Feng Zhang +10 more
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Journal ArticleDOI
Genome engineering using the CRISPR-Cas9 system
F. Ann Ran,Patrick D. Hsu,Jason Wright,Vineeta Agarwala,Vineeta Agarwala,David A. Scott,Feng Zhang +6 more
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Journal ArticleDOI
RNA-Guided Human Genome Engineering via Cas9
Prashant Mali,Luhan Yang,Kevin M. Esvelt,John Aach,Marc Güell,James E. DiCarlo,Julie E. Norville,George M. Church,George M. Church +8 more
TL;DR: The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
References
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Journal ArticleDOI
Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes
TL;DR: This work has identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs.
Journal ArticleDOI
CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3
Edze R. Westra,Paul B. G. van Erp,Tim Künne,Shi Pey Wong,Raymond H.J. Staals,Christel L.C. Seegers,Sander Bollen,Matthijs M. Jore,Ekaterina Semenova,Konstantin Severinov,Konstantin Severinov,Willem M. de Vos,Willem M. de Vos,Remus T. Dame,Renko de Vries,Stan J. J. Brouns,John van der Oost +16 more
TL;DR: It is shown that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM), which exclusively involves the crRNA-complementary DNA strand.
Journal ArticleDOI
Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems
TL;DR: Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISpr-cas systems but inactivated in Type I systems.
Journal ArticleDOI
RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions
Blake Wiedenheft,Esther van Duijn,Jelle B. Bultema,Sakharam P Waghmare,Kaihong Zhou,Arjan Barendregt,Wiebke Westphal,Albert J. R. Heck,Egbert J. Boekema,Mark J. Dickman,Jennifer A. Doudna +10 more
TL;DR: It is shown that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences.
Journal ArticleDOI
CRISPR-based adaptive immune systems
TL;DR: The current understanding of the remarkable CRISPR-Cas pathways is reviewed with particular attention to studies relevant to systems found in the archaea.
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