CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci

Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.

[1]  Yusuke Miyanari,et al.  Live visualization of chromatin dynamics with fluorescent TALEs , 2013, Nature Structural &Molecular Biology.

[2]  Wendell A. Lim,et al.  Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci , 2016, Nucleic acids research.

[3]  D. Peabody,et al.  RNA recognition site of PP7 coat protein. , 2002, Nucleic acids research.

[4]  Pedro P. Rocha,et al.  The origin of recurrent translocations in recombining lymphocytes: a balance between break frequency and nuclear proximity. , 2013, Current opinion in cell biology.

[5]  D. Gray,et al.  Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes , 2001, Nature Immunology.

[6]  M. Micsinai,et al.  Combined immunofluorescence and DNA FISH on 3D-preserved interphase nuclei to study changes in 3D nuclear organization. , 2013, Journal of visualized experiments : JoVE.

[7]  G. Almouzni,et al.  Mouse centric and pericentric satellite repeats form distinct functional heterochromatin , 2004, The Journal of cell biology.

[8]  Luke A. Gilbert,et al.  Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds , 2015, Cell.

[9]  A. Coulon,et al.  Kinetic competition during the transcription cycle results in stochastic RNA processing , 2014, eLife.

[10]  Heinrich Leonhardt,et al.  Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system , 2014, Nucleus.

[11]  B. Ren,et al.  The 3D genome in transcriptional regulation and pluripotency. , 2014, Cell stem cell.

[12]  Sigal Shachar,et al.  Identification of Gene Positioning Factors Using High-Throughput Imaging Mapping , 2015, Cell.

[13]  Yuval Kluger,et al.  Close proximity to Igh is a contributing factor to AID-mediated translocations. , 2012, Molecular cell.

[14]  Robert H. Singer,et al.  In the right place at the right time: visualizing and understanding mRNA localization , 2014, Nature Reviews Molecular Cell Biology.

[15]  Tom Misteli,et al.  Functional implications of genome topology , 2013, Nature Structural &Molecular Biology.

[16]  Pedro P. Rocha,et al.  Breaking TADs: insights into hierarchical genome organization. , 2015, Epigenomics.

[17]  Adam Burton,et al.  Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis , 2014, Nature Reviews Molecular Cell Biology.

[18]  Baohui Chen,et al.  Imaging genomic elements in living cells using CRISPR/Cas9. , 2014, Methods in enzymology.

[19]  G. Benson,et al.  Tandem repeats finder: a program to analyze DNA sequences. , 1999, Nucleic acids research.

[20]  T. Misteli,et al.  Spatial Dynamics of Chromosome Translocations in Living Cells , 2013, Science.

[21]  D. Heckl,et al.  Toward Whole-Transcriptome Editing with CRISPR-Cas9. , 2015, Molecular cell.

[22]  A S Belmont,et al.  In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition , 1996, The Journal of cell biology.

[23]  Carolyn A. Morrison,et al.  Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity , 2013, Nature Neuroscience.

[24]  Alexandro E. Trevino,et al.  Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex , 2014, Nature.

[25]  Jennifer E. Phillips-Cremins,et al.  Chromatin insulators: linking genome organization to cellular function. , 2013, Molecular cell.

[26]  Shaojie Zhang,et al.  Multicolor CRISPR labeling of chromosomal loci in human cells , 2015, Proceedings of the National Academy of Sciences.

[27]  Josée Dostie,et al.  An Overview of Genome Organization and How We Got There: from FISH to Hi-C , 2015, Microbiology and Molecular Reviews.

[28]  Yaojun Zhang,et al.  3D Trajectories Adopted by Coding and Regulatory DNA Elements: First-Passage Times for Genomic Interactions , 2014, Cell.