CRISPR RNA–guided activation of endogenous human genes
TL;DR: This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.
Abstract: Short guide RNAs (gRNAs) can direct catalytically inactive CRISPR-associated 9 nuclease (dCas9) to repress endogenous genes in bacteria and human cells. Here we show that single or multiple gRNAs can direct dCas9 fused to a VP64 transcriptional activation domain to increase expression of endogenous human genes. This proof-of-principle work shows that clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems can target heterologous effector domains to endogenous sites in human cells.
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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
TL;DR: In this paper, the authors describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions, and highlight challenges and future directions.
4,361 citations
TL;DR: This work shows that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells, and observes a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation.
Abstract: The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12 , as well as novel hits NF2 , CUL3 , TADA2B , and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
4,147 citations
01 Jun 2014
TL;DR: The development and applications of Cas9 are described for a variety of research or translational applications while highlighting challenges as well as future directions.
Abstract: Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.
3,270 citations
Cites background from "CRISPR RNA–guided activation of end..."
...By tiling a promoter with multiple sgRNAs, several groups have reported strong synergistic effects with nonlinear increases in activation (Perez-Pinera et al., 2013; Maeder et al., 2013b; Mali et al., 2013b)....
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...In general, targeting Cas9 activators with a single sgRNA to a particular endogenous gene promoter leads to only modest transcriptional upregulation (Konermann et al., 2013; Maeder et al., 2013b; Perez-Pinera et al., 2013; Mali et al., 2013b)....
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...Fusing them to modular, sequence-agnostic functional effector domains allows flexible recruitment of desired perturbations, such as transcriptional activation, to a locus of interest (Xu and Bestor, 1997; Beerli et al., 2000a; Konermann et al., 2013; Maeder et al., 2013a; Mendenhall et al., 2013)....
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...Previously, zinc finger proteins and TAL effectors have been used in a small number of proof-of-concept studies to achieve locus-specific targeting of epigenetic modifying enzymes (Beerli et al., 2000a; Konermann et al., 2013; Maeder et al., 2013a; Mendenhall et al., 2013)....
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TL;DR: A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells, which will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
Abstract: Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
2,930 citations
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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
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
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
TL;DR: This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale and can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects.
4,282 citations