SpCas9-sfGFP Nuclease

Product Introduction

SpCas9-sfGFP Nuclease is a fusion protein combining SpCas9 with superfolder GFP (sfGFP), designed to integrate gene editing functionality with fluorescent visualization capabilities. sfGFP is an engineered variant of traditional GFP. Through specific amino acid mutations, it exhibits enhanced protein folding efficiency and stability, enabling it to fold correctly and maintain intense fluorescence even when expressed as a fusion partner with other proteins. This overcomes the limitations of wild-type GFP, which is prone to misfolding or fluorescence quenching in fusion constructs.

This fusion protein is typically constructed by linking sfGFP to either the N-terminus or C-terminus of SpCas9 via a flexible polypeptide linker. Studies have demonstrated that the fusion of sfGFP does not interfere with the nuclease activity of SpCas9; the protein efficiently recognizes the NGG PAM sequence and introduces precise double-strand breaks at target DNA sites under the guidance of sgRNA. Concurrently, the fluorescent signal from sfGFP provides researchers with a direct, real-time tracking tool.

Utilizing SpCas9-sfGFP enables real-time monitoring of the spatiotemporal dynamics of the Cas9 protein at the live-cell level. Using fluorescence microscopy, the efficiency of Cas9 cellular entry, its transport within the cytoplasm, its translocation across the nuclear membrane, and its binding/unbinding kinetics at chromatin target sites within the nucleus can be visually observed. Combined with flow cytometry or high-throughput imaging analysis, the delivery efficiency of various systems (such as liposomes, viral particles, or nanomaterials) for Cas9 can be quantitatively assessed, providing a basis for optimizing gene therapy vector design. Furthermore, this fusion protein can be employed to screen for small molecule compounds affecting Cas9 activity or to study binding dynamics at specific genomic regions, thereby expanding the application boundaries of CRISPR technology in basic biology and translational medicine.