One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Cell (Impact Factor: 32.24). 08/2013; 154(6). DOI: 10.1016/j.cell.2013.08.022
Source: PubMed


The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

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Available from: Haoyi Wang, Dec 28, 2014
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    • "The expression level of the tagged protein is also adjustable to the endogenous level by inserting the tag into the endogenous gene locus by homologous recombination (Yang et al. 2013). Therefore, the changes in the protein localization can be tracked over time in single cells and also during development and differentiation in model systems. "
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    • "Similar to cultured human cells, most of the DSBs generated by Cas9 in 3PN zygotes were also repaired through NHEJ (Fig. 2A). ssDNA-mediated editing occurred only in 4 embryos (14.3%), and the edited embryos were mosaic, similar to findings in other model systems (Shen et al., 2013; Yang et al., 2013; Yen et al., 2014). Endogenous homologous sequences were also used as HDR templates, with an estimated editing efficiency of 25% (Fig. 2A "
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    ABSTRACT: Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
    Protein & Cell 04/2015; 6(5). DOI:10.1007/s13238-015-0153-5 · 3.25 Impact Factor
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    • "Unfortunately, there is thus far no genetic evidence for the participation of Dvl in axon guidance, due to lack of conditional Dvl alleles . The CRISPR/Cas system, which allows simultaneous targeting of multiple genes (Wang et al. , 2013, Yang et al. , 2013), could generate such in vivo evidence. In contrast, several in vitro experiments support such a role. "
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    ABSTRACT: The assembly of functional neuronal circuits depends on the correct wiring of axons and dendrites. To reach their targets, axons are guided by a variety of extracellular guidance cues, including Netrins, Ephrins, Semaphorins and Slits. Corresponding receptors in the growth cone, the dynamic structure at the tip of the growing axon, sense and integrate these positional signals, and activate downstream effectors to regulate cytoskeletal organization. In addition to the four canonical families of axon guidance cues mentioned above, some proteins that regulate planar cell polarity were recently found to be critical for axon guidance. The seven-transmembrane domain receptors Celsr3 and Fzd3, in particular, control the development of most longitudinal tracts in the central nervous system, and axon navigation in the peripheral, sympathetic and enteric nervous systems. Despite their unequivocally important role, however, underlying molecular mechanisms remain elusive. We do not know which extracellular ligands they recognize, whether they have co-receptors in the growth cone, and what their downstream effectors are. Here, we review some recent advances and discuss future trends in this emerging field. Copyright © 2015. Published by Elsevier Ltd.
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