Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Cell (Impact Factor: 32.24). 12/2013; 155(7):1479-91. DOI: 10.1016/j.cell.2013.12.001
Source: PubMed


The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

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    • "Efficiency of Cas9 - mediated genome engineering crucially depends on sgRNA efficacy , which varies widely ( summarized by Farboud and Meyer ( 2015 ) for C . elegans ) . To improve the stability of the sgRNA backbone , we synthesized a " flipped and extended " version ( Chen et al . 2013a ) , where an A - U basepair is flipped within the sgRNA stem - loop to disrupt a potential Pol III terminator and the stem - loop is extended by 5 bp . We placed this new backbone behind the K09B11 . 12 U6 promoter ( Friedland et al . 2013 ) to create a universal sgRNA cloning vector , pIK198 , which will be available through Add - gene"
    Dataset: Katic
    • "By supplying donor templates comprising exogenous sequence flanked by homology-containing stretches (commonly referred to as homology arms), the HDR pathway can be appropriated to make precise modifications including defined deletions, sequence substitutions, or insertions. Beyond the genome engineering applications of the CRISPR-Cas9 system, nuclease-dead Cas9 is being used as a sequence-specific repressor or activator of gene expression, and developed as a tool for probing genome structure and function without causing mutations (Bikard et al., 2013; Chen et al., 2013; Cheng et al., 2013; Fujita and Fujii, 2013; Gilbert et al., 2013; Maeder et al., 2013; Perez-Pinera et al., 2013; Qi et al., 2013; Anton et al., 2014; Kearns et al., 2014). "
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    ABSTRACT: The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 07/2015; 111:31.2.1-31.2.20. DOI:10.1002/0471142727.mb3102s111
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    • "Notably, the nonsense shRNAs and a reported efficient sgRNA targeting the mouse p53 gene (sgp53) (Xue et al., 2014) was adopted in the construct for a proof-of-concept experiment. The mature sgRNA derived from the miRsh-sgRNA cassette will have additive 7 nucleotides in the 5′-end (Fig. 1A), so we chose a reported optimized sgRNA backbone, the sgRNA (F+E) , to improve the mutagenesis efficiency (Chen et al., 2013), given that the sgRNAs with mispairing and addition in the 5′-end are still functional (Cong et al., 2013). "
    Protein & Cell 06/2015; 6(9). DOI:10.1007/s13238-015-0169-x · 3.25 Impact Factor
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