nCas9(D10A)-sfGFP Nickase

Product Introduction

nCas9(D10A)-sfGFP is a fusion protein combining a nickase variant, constructed by introducing the D10A point mutation into SpCas9, with superfolder GFP. It integrates precise DNA single-strand nicking capability with real-time visualization tracking. The D10A mutation specifically inactivates the RuvC nuclease domain while preserving the cleavage activity of the HNH domain. Consequently, guided by sgRNA, this enzyme cleaves only the DNA strand complementary to the target sequence, generating a precise single-strand nick, rather than the double-strand break produced by wild-type SpCas9. The fused sfGFP tag exhibits excellent folding efficiency and fluorescence stability, enabling real-time reporting of the protein's subcellular localization, dynamic distribution, and delivery efficiency at the live-cell level, providing a convenient optical tool for studying nickase behavior.

The unique value of nCas9 lies in its flexible application strategies. When used alone, the single-strand nick is primarily repaired with high fidelity through the intracellular base excision repair (BER) pathway, rarely resulting in random insertions or deletions. This makes it an ideal tool for studying DNA damage repair mechanisms or for scenarios requiring minimal genomic perturbation. Conversely, when employing a dual-nickase strategy, researchers can design a pair of sgRNAs targeting adjacent sites. These guide two nCas9 molecules to create nicks on opposite strands of the genomic target region, cooperatively forming a functional double-strand break. This approach retains gene editing efficiency comparable to wild-type Cas9, but because it requires simultaneous recognition and cooperation from two sgRNAs, tolerance for single off-target sites is extremely low. This significantly enhances editing specificity and reduces off-target risks. Consequently, this fusion protein is widely used in basic research and potential therapeutic applications requiring high-fidelity gene editing.