The CRISPR/Cas9 system for plant genome editing and beyond

doi:10.1016/J.BIOTECHADV.2014.12.006

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The CRISPR/Cas9 system for plant genome editing and beyond

Journal Article•DOI•

The CRISPR/Cas9 system for plant genome editing and beyond

Luisa Bortesi¹, Rainer Fischer¹•Institutions (1)

RWTH Aachen University¹

01 Jan 2015-Biotechnology Advances (Biotechnol Adv)-Vol. 33, Iss: 1, pp 41-52

TL;DR: The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (Cas9) system is described, a recently developed tool for the introduction of site-specific double-stranded DNA breaks and the strengths and weaknesses are highlighted.

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About: This article is published in Biotechnology Advances.The article was published on 2015-01-01 and is currently open access. It has received 948 citations till now. The article focuses on the topics: Transcription activator-like effector nuclease & Genome editing.

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Journal Article•DOI•

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

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Zhen Liang¹, Kunling Chen¹, Tingdong Li¹, Yi Zhang¹, Yanpeng Wang¹, Qian Zhao¹, Jinxing Liu¹, Huawei Zhang¹, Cuimin Liu¹, Yidong Ran, Caixia Gao¹ - Show less +7 more•Institutions (1)

Chinese Academy of Sciences¹

18 Jan 2017-Nature Communications

TL;DR: Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA, and the mutants obtained are completely transgene free.

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Abstract: Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

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652 citations

Journal Article•DOI•

Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology.

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Jeyabharathy Chandrasekaran, Marina Brumin, Dalia Wolf, Diana Leibman, Chen Klap, Mali Pearlsman, Amir Sherman, Tzahi Arazi, Amit Gal-On - Show less +5 more

01 Sep 2016-Molecular Plant Pathology

TL;DR: For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

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Abstract: Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

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594 citations

Journal Article•DOI•

Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease

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Tom Lawrenson¹, Oluwaseyi Shorinola¹, Nicola Stacey¹, Chengdao Li², Lars Østergaard¹, Nicola J. Patron¹, Cristobal Uauy¹, Wendy Harwood¹ - Show less +4 more•Institutions (2)

Norwich Research Park¹, Murdoch University²

30 Nov 2015-Genome Biology

TL;DR: The use of RNA-guided Cas9 is demonstrated to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species.

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Abstract: The RNA-guided Cas9 system represents a flexible approach for genome editing in plants. This method can create specific mutations that knock-out or alter target gene function. It provides a valuable tool for plant research and offers opportunities for crop improvement. We investigate the use and target specificity requirements of RNA-guided Cas9 genome editing in barley (Hordeum vulgare) and Brassica oleracea by targeting multicopy genes. In barley, we target two copies of HvPM19 and observe Cas9-induced mutations in the first generation of 23 % and 10 % of the lines, respectively. In B. oleracea, targeting of BolC.GA4.a leads to Cas9-induced mutations in 10 % of first generation plants screened. In addition, a phenotypic screen identifies T0 plants with the expected dwarf phenotype associated with knock-out of the target gene. In both barley and B. oleracea stable Cas9-induced mutations are transmitted to T2 plants independently of the T-DNA construct. We observe off-target activity in both species, despite the presence of at least one mismatch between the single guide RNA and the non-target gene sequences. In barley, a transgene-free plant has concurrent mutations in the target and non-target copies of HvPM19. We demonstrate the use of RNA-guided Cas9 to generate mutations in target genes of both barley and B. oleracea and show stable transmission of these mutations thus establishing the potential for rapid characterisation of gene function in these species. In addition, the off-target effects reported offer both potential difficulties and specific opportunities to target members of multigene families in crops.

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427 citations

Cites result from "The CRISPR/Cas9 system for plant ge..."

...These mutation frequencies are comparable to those reported in stable T0 transformants from other monocotyledonous species such as wheat [12], rice (Oryza sativa; reviewed in [17]) and sorghum (Sorghum bicolor; [18])....
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Journal Article•DOI•

The heat‐shock protein/chaperone network and multiple stress resistance

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Pierre Jacob, Heribert Hirt¹, Abdelhafid Bendahmane•Institutions (1)

King Abdullah University of Science and Technology¹

23 Feb 2017-Plant Biotechnology Journal

TL;DR: The role of HSPs/chaperones in regulating diverse signalling pathways is illustrated and several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/ chaperone network are discussed.

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Abstract: Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network.

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421 citations

Cites background from "The CRISPR/Cas9 system for plant ge..."

...In this regard, genome editing by CRISPR-CAS9 would be cleaner and faster (Bortesi and Fischer, 2015)....
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Journal Article•DOI•

Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts.

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Mariette Andersson¹, Helle Turesson¹, Alessandro Nicolia², Ann Sofie Fält¹, Mathias Samuelsson, Per Hofvander¹ - Show less +2 more•Institutions (2)

Swedish University of Agricultural Sciences¹, Biotec²

01 Jan 2017-Plant Cell Reports

TL;DR: Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential.

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Abstract: Key message Altered starch quality with full knockout ofGBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts.

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369 citations

Cites background or methods or result from "The CRISPR/Cas9 system for plant ge..."

...2015); in comparison to the more generally used DNA sequencing method (Bortesi and Fischer 2015) it has a much shorter time span from sampling to identification of mutants....
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...U6 promoters have been the most commonly used type of promoter for driving expression of sgRNA in plants (Bortesi and Fischer 2015)....
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...Detection of indels using PCR and capillary electrophoresis is used here and elsewhere (Ramlee et al. 2015; Yang et al. 2015); in comparison to the more generally used DNA sequencing method (Bortesi and Fischer 2015) it has a much shorter time span from sampling to identification of mutants....
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References

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Journal Article•DOI•

A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

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Martin Jinek¹, Krzysztof Chylinski², Krzysztof Chylinski³, Ines Fonfara², Michael H. Hauer¹, Jennifer A. Doudna, Emmanuelle Charpentier² - Show less +3 more•Institutions (3)

University of California, Berkeley¹, Umeå University², Max F. Perutz Laboratories³

17 Aug 2012-Science

TL;DR: 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.

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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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12,865 citations

"The CRISPR/Cas9 system for plant ge..." refers background or methods in this paper

...A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5′-NGG-3′ (Gasiunas et al., 2012; Jinek et al., 2012) but less frequently NAG (Hsu et al., 2013)....
[...]
...A prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5′-NGG-3′ (Gasiunas et al., 2012; Jinek et al., 2012) but less frequently NAG (Hsu et al....
[...]
...Although a 20 nt sequence in the gRNA was initially considered necessary to determine specificity, it was later shown that only the 8–12 nt at the 3′ end (the seed sequence) is needed for target site recognition and cleavage (Cong et al., 2013; Jiang et al., 2013a; Jinek et al., 2012), whereas multiple mismatches in the PAM-distal region can be tolerated, depending on the total number and arrangement (Fu et al....
[...]
...…that only the 8–12 nt at the 3′ end (the seed sequence) is needed for target site recognition and cleavage (Cong et al., 2013; Jiang et al., 2013a; Jinek et al., 2012), whereas multiple mismatches in the PAM-distal region can be tolerated, depending on the total number and arrangement (Fu et…...
[...]
...…could be reprogrammed simply by changing 20 nucleotides in the crRNA and that the targeting specificity of the crRNA could be combinedwith the structural properties of the tracrRNA in a chimeric single guide RNA (gRNA), thus reducing the system from three to two components (Jinek et al., 2012; Fig....
[...]

Journal Article•DOI•

Multiplex Genome Engineering Using CRISPR/Cas Systems

[...]

Le Cong¹, Le Cong², F. Ann Ran¹, F. Ann Ran², David M. Cox¹, David M. Cox², Shuailiang Lin², Shuailiang Lin³, Robert P. J. Barretto⁴, Naomi Habib², Patrick D. Hsu¹, Patrick D. Hsu², Xuebing Wu², Wenyan Jiang⁵, Luciano A. Marraffini⁵, Feng Zhang² - Show less +12 more•Institutions (5)

Harvard University¹, Massachusetts Institute of Technology², Tsinghua University³, Columbia University⁴, Rockefeller University⁵

15 Feb 2013-Science

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.

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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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12,265 citations

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 - Show less +7 more

01 Feb 2013

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.

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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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10,746 citations

"The CRISPR/Cas9 system for plant ge..." refers background or result in this paper

...Importantly, it was also shown that multiple gRNAs with different sequences could be used to achieve high-efficiency multiplex genome engineering at different loci simultaneously (Cong et al., 2013; Mali et al., 2013)....
[...]
...…that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013)....
[...]
...Shortly thereafter, five independent groups demonstrated that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013)....
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...In terms of comparative performance, the first studies using the CRISPR/Cas9 system for genome editing in mammalian cell lines and zebrafish embryos showed that the technology was at least as efficient as ZFNs and TALENs targeting the same sites (Cong et al., 2013; Hwang et al., 2013) and in some cases even higher (Ding et al....
[...]
...…the first studies using the CRISPR/Cas9 system for genome editing in mammalian cell lines and zebrafish embryos showed that the technology was at least as efficient as ZFNs and TALENs targeting the same sites (Cong et al., 2013; Hwang et al., 2013) and in some cases even higher (Ding et al., 2013)....
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Journal Article•DOI•

RNA-Guided Human Genome Engineering via Cas9

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Prashant Mali¹, Luhan Yang¹, Kevin M. Esvelt², John Aach¹, Marc Güell¹, James E. DiCarlo³, Julie E. Norville¹, George M. Church², George M. Church¹ - Show less +5 more•Institutions (3)

Harvard University¹, Wyss Institute for Biologically Inspired Engineering², Boston University³

15 Feb 2013-Science

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.

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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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8,197 citations

"The CRISPR/Cas9 system for plant ge..." refers background or result in this paper

...Importantly, it was also shown that multiple gRNAs with different sequences could be used to achieve high-efficiency multiplex genome engineering at different loci simultaneously (Cong et al., 2013; Mali et al., 2013)....
[...]
...…that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013)....
[...]
...Shortly thereafter, five independent groups demonstrated that the two-component system was functional in eukaryotes (human, mouse and zebrafish), indicating that the other functions of the CRISPR locus genes were supported by endogenous eukaryotic enzymes (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jinek et al., 2013; Mali et al., 2013)....
[...]
...…genome modifications with efficiencies comparable to the unmodified enzyme, butwith 50- to 1500-fold greater specificity in human and mouse cells (Cho et al., 2014; Duda et al., 2014;Mali et al., 2013; Ran et al., 2013; Shen et al., 2014) and Arabidopsis (Fauser et al., 2014; Schiml et al., 2014)....
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...This strategy can yield precise genome modifications with efficiencies comparable to the unmodified enzyme, butwith 50- to 1500-fold greater specificity in human and mouse cells (Cho et al., 2014; Duda et al., 2014;Mali et al., 2013; Ran et al., 2013; Shen et al., 2014) and Arabidopsis (Fauser et al....
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Journal Article•DOI•

CRISPR provides acquired resistance against viruses in prokaryotes

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Rodolphe Barrangou¹, Christophe Fremaux, Hélène Deveau², Melissa Richards¹, Patrick Boyaval, Sylvain Moineau², Dennis A. Romero¹, Philippe Horvath - Show less +4 more•Institutions (2)

Danisco¹, Laval University²

23 Mar 2007-Science

TL;DR: It is found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences, and CRISPR provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.

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Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.

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5,045 citations

"The CRISPR/Cas9 system for plant ge..." refers background in this paper

...The CRISPR arrays, including the spacers, are transcribed during subsequent encounters with invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs) approximately 40 nt in length, which combine with the transactivating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease (Barrangou et al., 2007)....
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...This cleaves homologous double-stranded DNA sequences known as protospacers in the invading DNA (Barrangou et al., 2007)....
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...…the spacers, are transcribed during subsequent encounters with invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs) approximately 40 nt in length, which combine with the transactivating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease (Barrangou et al., 2007)....
[...]
...Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007)....
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