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Journal ArticleDOI

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease

TL;DR: A bacterial CRISPR RNA/Cas9 system is adapted to precisely engineer the Drosophila genome and it is reported that Cas9-mediated genomic modifications are efficiently transmitted through the germline.
Abstract: We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
Citations
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Journal ArticleDOI
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.
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.

8,663 citations

Journal ArticleDOI
28 Nov 2014-Science
TL;DR: The power of the CRISPR-Cas9 technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders is illustrated.
Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

4,774 citations


Cites background from "Genome engineering of Drosophila wi..."

  • ...Efficient genome engineering to allow targeted genome modifications in the germ lines of animal models such as fruit flies (113, 114), zebrafish (94, 115), nematodes (116), salamanders (117), and frogs (118, 119) is now possible with the development of the CRISPR-Cas9 technology....

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Journal ArticleDOI
TL;DR: In this article, the Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing.
Abstract: The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

4,113 citations

01 Sep 2013
TL;DR: It is found that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches.
Abstract: The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

3,421 citations

Journal ArticleDOI
18 Jul 2013-Cell
TL;DR: The results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISpri as a general tool for the precise regulation of gene expression in eukaryotic cells.

3,165 citations

References
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Journal ArticleDOI
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.
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.

12,865 citations


"Genome engineering of Drosophila wi..." refers background in this paper

  • ...All plasmids used in this study will be made available to academic laboratories through the nonprofit distributor Addgene along with detailed guidance at http://FlyCRISPR.molbio.wisc.edu....

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  • ...…sequence PAM cut site                                                                                 Figure  S1      CRISPR  RNA/Cas9  molecular  reagents  used  in  this  study     (A)  Schematic  of  the  modified  CRISPR  RNA/Cas9…...

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  • ...Moreover, it may be possible to bias repair to HR over NHEJ by exploiting a Cas9 mutant that induces single-strand DNA breaks (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013) or conducting Cas9-mediated genome engineering in a genetic Note 1033 background that reduces NHEJ (Beumer et al.…...

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  • ...Recognizing the potential of harnessing this system for precise genome engineering in other organisms, Jinek and colleagues (Jinek et al. 2012) identified a minimal twocomponent system required for the site-specific cleavage of DNA—Cas9 and a chimeric RNA (chiRNA) comprising the crRNA and tracrRNA…...

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  • ...Cleavage also requires that the 39 end of the genomic target sequence contains a 3-basepair (bp) proto-spacer adjacent motif (PAM) sequence, NGG, which differentiates self from invading DNA in the endogenous system (Jinek et al. 2012)....

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Journal ArticleDOI
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.
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.

12,265 citations

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.
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.

10,746 citations

Journal ArticleDOI
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.
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.

8,197 citations


"Genome engineering of Drosophila wi..." refers background in this paper

  • ...…findings from mammalian cell culture in which 19- and 118-bp sequences were deleted by cotransfection of Cas9 and two chiRNAs (Cong et al. 2013; Mali et al. 2013) and demonstrate that such “multiplex” genome editing is an effective tool for precisely deleting large genomic regions in organisms....

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  • ...…it may be possible to bias repair to HR over NHEJ by exploiting a Cas9 mutant that induces single-strand DNA breaks (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013) or conducting Cas9-mediated genome engineering in a genetic Note 1033 background that reduces NHEJ (Beumer et al. 2008)....

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  • ...E-mail: oconnorgiles@wisc.edu Genetics, Vol. 194, 1029–1035 August 2013 1029 eukaryotic genomes, including cultured mammalian cells, human stem cells, and yeast (Cho et al. 2013; Cong et al. 2013; Dicarlo et al. 2013; Ding et al. 2013; Jinek et al. 2013; Mali et al. 2013)....

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Journal ArticleDOI
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.
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.

5,045 citations


"Genome engineering of Drosophila wi..." refers background in this paper

  • ...CRISPRs (clustered regularly interspaced short palindromic repeats) were first identified in Escherichea coli in 1987 and later shown to function as part of an adaptive immune system in bacteria and archaea (Ishino et al. 1987; Makarova et al. 2006; Barrangou et al. 2007)....

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