Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

Encell, Lance P.; Ohana, Rachel Friedman; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G.; Los, Georgyi V.; McDougall, Mark G.; Zimprich, Chad; Karassina, Natasha; Learish, Randall D.; Hurst, Robin; Hartnett, James R.; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Méndez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L.; Slater, Michael; Urh, Marjeta; Darzins, Al; Klaubert, Dieter H.; Bulleit, Robert F.; Wood, Keith V.

doi:10.2174/1875397301206010055

Cited by 120 publications

(129 citation statements)

References 80 publications

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“…The Halo tag can be labeled efficiently and covalently by Halo ligands conjugated to a variety of organic fluorescent dyes for different imaging purposes (10). We purified recombinant dCas9 fusion protein expressed in Escherichia coli and labeled the protein with Halo ligands conjugated with Janelia Fluor 646 (JF646) (11).…”

Section: Resultsmentioning

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.

249

214

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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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Section: Resultsmentioning

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.

249

214

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“…To increase its stability, 22 mutations have previously been introduced to create the more stable HaloTag7 protein for recombinant protein work 15; 16 . We hypothesized that these mutations would also provide increased stability in mammalian cells.…”

Section: Resultsmentioning

confidence: 99%

A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

et al. 2013

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Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.

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“…Similarly, mutagenesis of small single-chain enzymes has yielded variants that self-label covalently with a fluorophore substituent in the course of one catalytic turnover when provided with an appropriate cell-permeable, dye-derivatized substrate. This approach produced the so-called SNAP (Corrêa et al 2013), CLIP (Gautier et al 2008), and Halo (Encell et al 2012) tags. It is important to emphasize, however, that studies tracking hybrid proteins must be confirmed by independent approaches (e.g., genetic complementation) that show they retain biological function; sadly, such constructs are often used and conclusions drawn without this necessary validation.…”

Section: Seeing Is Believingmentioning

confidence: 99%

Signal Transduction: From the Atomic Age to the Post-Genomic Era

Thorner

Hunter

Cantley

et al. 2014

Cold Spring Harbor Perspectives in Biology

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SUMMARY We have come a long way in the 55 years since Edmond Fischer and the late Edwin Krebs discovered that the activity of glycogen phosphorylase is regulated by reversible protein phosphorylation. Many of the fundamental molecular mechanisms that operate in biological signaling have since been characterized and the vast web of interconnected pathways that make up the cellular signaling network has been mapped in considerable detail. Nonetheless, it is important to consider how fast this field is still moving and the issues at the current boundaries of our understanding. One must also appreciate what experimental strategies have allowed us to attain our present level of knowledge. We summarize here some key issues (both conceptual and methodological), raise unresolved questions, discuss potential pitfalls, and highlight areas in which our understanding is still rudimentary. We hope these wide-ranging ruminations will be useful to investigators who carry studies of signal transduction forward during the rest of the 21st century.

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Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

Cited by 120 publications

References 80 publications

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

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

A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

Signal Transduction: From the Atomic Age to the Post-Genomic Era

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