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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    • "To implement scCRISPR, we designed self-complementary palindromic sgRNA plasmids (sgPals) that should induce their own cleavage after complexing with Cas9 in cells. We used the improved ''FE'' sgRNA design that has been shown to increase Cas9 cleavage efficiency (Chen et al., 2013). To minimize off-target genomic DNA cleavage by sgPal, we designed an sgPal sequence with minimal predicted off-target cleavage potential (see the Supplemental Experimental Procedures). "
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    ABSTRACT: We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%-4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
    Stem Cell Reports 11/2015; DOI:10.1016/j.stemcr.2015.09.022 · 5.37 Impact Factor
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    • "In another strategy the introduction of an A-U base pair to the gRNA scaffold, stabilizes the gRNA scaffold. Besides, extension of the base pair in the hairpin increases the binding rate of gRNA to target site and finally enhances its on-target specificity (Chen et al., 2013) (shown in Figure 2D). "
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    Current issues in molecular biology 10/2015; 20:1-12. · 5.75 Impact Factor
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    • "Gene tagging dCas9 GFP / SunTag Any Immunostaining Microscopy Western blot Tanenbaum et al . , 2014 Genome locus visualization dCas9 BFP EGFP GFP , 3xGFP , mCherry Any Microscopy Chen et al . , 2013 ; Ma et al . , 2015"
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    ABSTRACT: CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.
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