dCas9-GFP Protein

Product Introduction

dCas9-GFP is a versatile imaging tool formed by fusing Green Fluorescent Protein (GFP) to nuclease-dead Cas9 (dCas9), enabling real-time visualization and tracking of genomic loci. This fusion protein utilizes dCas9 as its foundational backbone, which harbors D10A and N863A double mutations that completely abolish endonuclease activity while fully retaining the ability to target and bind DNA specifically guided by a single-guide RNA (sgRNA). The GFP tag provides a stable and bright fluorescent signal, allowing researchers to directly observe the position and dynamics of specific genomic sequences in living cells.

The core value of dCas9-GFP lies in transforming "invisible" genetic loci into "visible" fluorescent spots. Upon expression of dCas9-GFP and a specific sgRNA, the fusion protein precisely binds to the target DNA region, appearing as distinct nuclear foci under a fluorescence microscope. This technique overcomes the limitation of traditional methods like FISH, which are restricted to fixed cells, making it possible to track the dynamic behavior of chromatin in living cells, including telomere dynamics, spatial positional changes of loci during the cell cycle, and chromatin interactions.

In the study "TriTag: an integrative tool to monitor chromatin dynamics and gene expression in living cells," dCas9-GFP assumes an even more significant role—as the core sensor responsible for DNA visualization within the TriTag system. In this multicolor, multimodality live-cell imaging platform, dCas9-GFP is no longer an imaging tool working in isolation but operates synergistically with other fluorescent modules tracking transcriptional activity and protein expression. This enables the direct correlation analysis of the spatial dynamics of specific chromatin loci with their transcriptional status and translational products, providing an unprecedented integrative perspective for studying gene regulatory mechanisms.

Product Specifications

Product Specifications

Application Scenarios

Live-Cell Dynamic Imaging:

Real-time tracking of the position and movement trajectories of specific genes within the nucleus under a microscope.

Observing the three-dimensional structure of chromosomes or labeling telomere length changes. While traditional FISH technology is limited to post-fixation staining, dCas9 enables the visualization of live cellular processes.

Application in the TriTag System:

Enabled discoveries regarding the relationship between chromatin dynamics and transcriptional regulation through observation of dCas9-GFP signal changes.

References

Gilbert LA, et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 154(2):442-451.

Perez-Pinera P, et al. (2013). RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 10(10):973-976.

Chavez A, et al. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 12(4):326-328.

Ramu G, et al. (2018). Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. Nucleic Acids Res. 46(12):e71.

Xu H, et al. (2020). TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells. Nucleic Acids Res. 48(22):13013-13014.