Article

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: 31.48). 01/2013; 339(6121). DOI: 10.1126/science.1231143
Source: PubMed

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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    • "The plasmid pTOXO_Cas9-CRISPR was synthesized by GenScript in a pUC57 plasmid backbone. This vector harbors two expression units: a C-terminal HA-NLS -YFP-NLS tagged Cas9 gene from Streptococcus pyogenes (Cong et al., 2013) under the control of the Toxoplasma TUB8 promoter and the TgU6 promoter upstream of the single guide RNA (sgRNA) cloning site (BsaI) fused to the Cas9 recognition sequence. The 20-mer oligos corresponding to the This article is protected by copyright. "
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    ABSTRACT: Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into in the infected cells in order to interfere with host-signaling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N-terminal region. Because the first element contains a putative PEXEL linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N- and C-terminal epitope tags, we provide evidence for protein proteolysis occurring in the N-terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium Plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a PEXEL-dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, though the GRA24 maturation mechanism is TgASP5-independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Cellular Microbiology 08/2015; DOI:10.1111/cmi.12498 · 4.82 Impact Factor
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    • "First, we tested U6 promoters as a means of expressing gRNAs. U6 snRNAs are transcribed by RNA polymerase III, which is suitable for expressing small RNAs with precisely defined 5′ and 3′ ends, such as gRNAs (Cong et al., 2013; Dickinson et al., 2013; Jinek et al., 2013; Mali et al., 2013b; Ren et al., 2013). U6 promoters generate transcripts that start with a G and can thus be used to optimally target sequences that conform to G-N 16-19 -NGG (in practice, a mismatched G at the 5′ end of the gRNA can be tolerated; Fu et al., 2014; Hwang et al., 2013a). "
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    ABSTRACT: Gene editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knock-out and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knock-outs; 71-100% carry such mutations in their germline and transmit them to the next generation. We show that CRISPR-mediated gene knock-out of the Tribolium E-cadherin gene gives defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marked transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs. © 2015. Published by The Company of Biologists Ltd.
    Development 07/2015; DOI:10.1242/dev.125054 · 6.27 Impact Factor
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    • "Please cite this article in press as: Zhong et al., CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library, Cell Stem Cell (2015), http://dx.doi.org/10.1016/j.stem.2015.06.005 Lentiviral Infection of Haploid Cells The lenti-sgRNA library and lenti-Cas9 plasmids were reported previously (Cong et al., 2013; Koike-Yusa et al., 2014) and provided by Addgene. For preparation of the lentivirus, the HEK293T cells in a 10-cm dish were transfected with 3 mg of a lentiviral vector (lenti-sgRNA library or lenti-Cas9) and 9 mg of Vira- Power Lentiviral Packaging Mix (Invitrogen) by Lipofectamine 2000 Reagent (Invitrogen) in accordance with the manual. "
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    ABSTRACT: Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell stem cell 07/2015; 17(2). DOI:10.1016/j.stem.2015.06.005 · 22.15 Impact Factor
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