Multiplex genome engineering using CRISPR/Cas systems

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA and McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science (Impact Factor: 33.61). 01/2013; 339(6121). DOI: 10.1126/science.1231143
Source: PubMed


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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Available from: Le Cong, Mar 24, 2015
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    • "However, our successful design of five stable CTD variants revealed the high plasticity of the mammalian CTD and confirmed that substantial manipulation of CTD is possible (Bartolomei et al., 1988).Consequently, our established CTD variants are a powerful tool to gain deeper insights into CTD heptad-specific phosphorylation after the knock out of cellular kinases or phosphatases. For example, by establishing specific CTD kinase analog sensitive mammalian cell lines (Bartkowiak et al., 2015;Laitem et al., 2015) using the genomic engineering tool CRISPR/Cas (Cong et al., 2013;Fu et al., 2013;Mali et al., 2013), CTD-specific kinase knockdowns will possibly link different CTD subregions to specific kinase activities. In the future, it may also allow us to monitor changes in CTD phosphorylation after activation of specific signaling pathways. "
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    ABSTRACT: The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
    Full-text · Article · Jan 2016 · Molecular Cell
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    • "The high target specificity of Cas9 nickase was further verified by Skarnes' group. They found Figure 1The timeline for applications of CRISPR/Cas technology in model organisms[28]Th loci N2A Feb, 2013[28]Note: ZFN, zinc finger nuclease; TALEN, transcription activator-like effector nuclease; CRISPR, clustered regularly-interspaced short palindromic repeat; Cas, CRISPR-associated; NTDF3, neurotrophin-3; CCR5, chemokine (C–C motif) receptor 5; AAVS1, adeno-associated virus integration site 1; EMX1, empty spiracles homeobox 1; Th, tyrosine hydroxylase; gol, golden; tnikb, TRAF2 and NCK-interacting protein kinase; tia1, T-cellrestricted intracellular antigen-1; gsk3b, glycogen synthase kinase 3 beta. no detectable NHEJ-induced damage at the reported off-target sites recognized by wild-type Cas9 endonuclease both in mouse embryos and cultured cells[58]. "
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    ABSTRACT: Technological advances are important for innovative biological research. Development of molecular tools for DNA manipulation, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas), has revolutionized genome editing. These approaches can be used to develop potential therapeutic strategies to effectively treat heritable diseases. In the last few years, substantial progress has been made in CRISPR/Cas technology, including technical improvements and wide application in many model systems. This review describes recent advancements in genome editing with a particular focus on CRISPR/Cas, covering the underlying principles, technological optimization, and its application in zebrafish and other model organisms, disease modeling, and gene therapy used for personalized medicine.
    Full-text · Article · Jan 2016
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    • "s9 system lies in is its simplicity and flexibility . Whereas RNAi development can be time consuming and / or costly , often requiring many iterations before producing an acceptable level of knockdown ( Moore et al . , 2010 ) , nearly every targeting sequence designed for use with CRISPR / Cas9 has a high chance of producing the desired knockout ( Cong et al . , 2013 ; Mali et al . , 2013 ) . This efficiency allows for rapid and effective switching between gene targets and importantly , for developing multiple targeting sequences to edit multiple genes simultaneously ."
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    ABSTRACT: Gene editing tools are essential for uncovering how genes mediate normal brain–behavior relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene editing technology now allows neuroscientists unprecedented access to edit the genome efficiently. Although many important tools have been developed, here we focus on approaches that allow for rapid gene editing in the adult nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques. CRISPR/Cas9 is a flexible gene editing tool, allowing the genome to be manipulated in diverse ways. For instance, CRISPR/Cas9 has been successfully used to knockout genes, knock-in mutations, overexpress or inhibit gene activity, and provide scaffolding for recruiting specific epigenetic regulators to individual genes and gene regions. Moreover, the CRISPR/Cas9 system may be modified to target multiple genes at one time, affording simultaneous inhibition and overexpression of distinct genetic targets. Although many of the more advanced applications of CRISPR/Cas9 have not been applied to the nervous system, the toolbox is widely accessible, such that it is poised to help advance neuroscience. Anti-sense nucleotide-based technologies can be used to rapidly knockdown genes in the brain. The main advantage of anti-sense based tools is their simplicity, allowing for rapid gene delivery with minimal technical expertise. Here, we describe the main applications and functions of each of these systems with an emphasis on their many potential applications in neuroscience laboratories.
    Full-text · Article · Jan 2016 · Frontiers in Genetics
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