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CRISPR

About: CRISPR is a(n) research topic. Over the lifetime, 13577 publication(s) have been published within this topic receiving 603858 citation(s). The topic is also known as: clustered regularly interspaced short palindromic repeats.

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Open accessJournal ArticleDOI: 10.1126/SCIENCE.1231143
Le Cong1, Le Cong2, F. Ann Ran1, F. Ann Ran2  +12 moreInstitutions (5)
15 Feb 2013-Science
Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. 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. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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Topics: CRISPR/Cpf1 (72%), CRISPR (66%), Transcription activator-like effector nuclease (66%) ...read more

10,364 Citations


Open accessJournal ArticleDOI: 10.1126/SCIENCE.1225829
17 Aug 2012-Science
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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Topics: Trans-activating crRNA (78%), CRISPR/Cpf1 (64%), Cas9 (63%) ...read more

10,148 Citations


Open access
01 Feb 2013-
Abstract: Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. 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. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

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Topics: CRISPR (58%), Genome engineering (53%), Multiplex (51%)

9,336 Citations


Open accessJournal ArticleDOI: 10.1126/SCIENCE.1232033
Prashant Mali1, Luhan Yang1, Kevin M. Esvelt2, John Aach1  +5 moreInstitutions (3)
15 Feb 2013-Science
Abstract: Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

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Topics: CRISPR/Cpf1 (68%), CRISPR interference (64%), CRISPR (64%) ...read more

7,430 Citations


Open accessJournal ArticleDOI: 10.1038/NPROT.2013.143
F. Ann Ran, Patrick D. Hsu, Jason Wright1, Vineeta Agarwala1  +3 moreInstitutions (2)
01 Nov 2013-Nature Protocols
Abstract: Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe 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. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

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Topics: Genome editing (63%), CRISPR (60%), Cas9 (60%) ...read more

6,858 Citations


Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202231
20212,110
20202,152
20192,126
20181,936
20171,740

Top Attributes

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Topic's top 5 most impactful authors

Feng Zhang

149 papers, 87.6K citations

Jennifer A. Doudna

129 papers, 40.6K citations

Rodolphe Barrangou

106 papers, 16.3K citations

Eugene V. Koonin

65 papers, 13.1K citations

Luciano A. Marraffini

54 papers, 32.4K citations

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