A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

Tanenbaum, Marvin E.; Gilbert, Luke A.; Qi, Lei S.; Weissman, Jonathan S.; Vale, Ronald D.

doi:10.1016/j.cell.2014.09.039

Cited by 1,256 publications

(1,223 citation statements)

References 40 publications

(69 reference statements)

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“…Based on our previous TALEColor study (7) and other considerations, we estimate that 150-200 FP molecules need to be stationed on a given chromosomal site to create a detectable signal (i.e., ∼50-70 dCas9-FP/sgRNA complexes when using 3XGFP). At the time that this manuscript was submitted for publication, a protein-tagging system (SunTag) was reported in which a repeating peptide array can recruit up to 24 copies of GFP (19). If successfully deployed, this would significantly enhance the CRISPR/dCas9 chromosome labeling signals and extend the detection limit.…”

Section: Discussionmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

361

338

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The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.4D nucleome | telomeres | pericentromeric DNA | chromosomes

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Section: Discussionmentioning

confidence: 99%

Multicolor CRISPR labeling of chromosomal loci in human cells

Naseri

Reyes-Gutierrez

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

361

338

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“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Guan

et al. 2015

Journal of General Virology

178

132

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CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5 D32 variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4 + T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4 + Tlymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4 + T-cells utilizing adenovirus-delivered CRISPR/Cas9.

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“…The type II clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated caspase 9 (Cas9) system derived from Streptococcus pyogenes has become a revolutionary tool for targeted genome editing (4), and its nuclease-deficient derivatives (dCas9) also are used for control of gene expression (5) and visualization of genomic loci in live cells (6)(7)(8) through fusion with a transcription-regulation domain or a fluorescent protein, respectively. Inherent multiplexing features offered by the CRISPR system hold great promise for applications in highthroughput assays.…”

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confidence: 99%

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

Deng

Shi

Tjian

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

238

204

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Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

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A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

Cited by 1,256 publications

References 40 publications

Multicolor CRISPR labeling of chromosomal loci in human cells

Multicolor CRISPR labeling of chromosomal loci in human cells

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

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