A protein tagging system for signal amplification in gene expression and fluorescence imaging

By M. Tanenbaum, Luke A. Gilbert, Lei S. Qi, Jonathan S. Weissman, R. Vale. Cell, 2014

1,449 citations recorded by the source corpus.

Summary Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a novel protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and in controlling biological outputs.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/f163eb8260c03f29a28dab485d9562bf650a9f47

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Citations in the corpus

CRISPR/Cas Systems towards Next-Generation Biosensing. (2019, methodology): “Later, by using dCas9-SunTag or an sgRNA scaffold with multiple protein binding sites, more fluorescent proteins can be recruited to a single locus to enhance the fluorescent signals [42,43].”
Single-Cell and Single-Molecule Analysis of Gene Expression Regulation (2016, background): “IF ( green) recognizes the scFv-sfGFP bound to the 24 copies of the GCN4 sequence (SunTag system). smFISH (red ) recognizes the transcript coding for the GCN4 polypeptide.”
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation (2014, methodology): “We recently developed an improved CRISPRa method, termed sunCas9, in which expression of a single sgRNAwith one binding site is sufficient to robustly activate transcription (Tanenbaum et al., 2014, this issue).”
The Biogenesis, Functions, and Challenges of Circular RNAs. (2018, neutral): “For instance, cellular starvation led to augmented circMbl translation (Pamudurti et al., 2017), and heat shock promoted the translation of GFP from the m6A-containing circRNA plasmid (Yang et al., 2017).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “Shortly after reporting the gene interference CRISPR variant, this same group developed a gene expression/amplification CRISPR (CRISPR activation, CRISPRa) by creating a fusion protein of a dCas9 and a repeating peptide array transcription factor (Tanenbaum et al., 2014).”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “All of these studies show that synergistically recruiting multiple activators to the dCas9 target locus enhances the activation of the CRISPRa system (180, 184, 185).”
TrackMate: An open and extensible platform for single-particle tracking. (2017, methodology): “TrackMate has been used in protein motility studies [40,41], molecular motor tracking [42], axonal transport [43], sperm cell tracking [44], Golgi bodies tracking [45] in plants, wound healing [46], bacterial bioﬁlm micro-rheology [47], cell movement on stiffness-patterned substrate [48], cell…”