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Cat. No.: NCAS9SG-500 (for 500pmol)

Cat. No.: NCAS9SG-2500 (for 2500pmol)

Description

nCas9(D10A)-sfGFP is a fusion protein consisting of the SpCas9 nickase variant carrying the D10A point mutation and superfolder GFP (sfGFP). This design integrates precise single‑strand DNA cleavage capability with robust real‑time fluorescence visualization. The D10A mutation specifically inactivates the RuvC nuclease domain while preserving the catalytic activity of the HNH domain. As a result, the enzyme introduces a single‑strand nick exclusively on the DNA strand complementary to the sgRNA, rather than generating a double‑strand break as seen with wild‑type SpCas9.

The sfGFP tag provides enhanced folding efficiency and fluorescence stability, enabling reliable live‑cell tracking of the protein’s subcellular localization, dynamic behavior, and delivery efficiency. This makes nCas9(D10A)-sfGFP a convenient optical tool for studying nickase activity in real time.

The unique value of nCas9 lies in its flexible application strategies. When used alone, the single‑strand nick is predominantly repaired through the high‑fidelity base excision repair pathway, producing minimal indels. This makes the enzyme ideal for studying DNA damage repair mechanisms or applications requiring minimal genomic perturbation.

In contrast, when used in a paired‑nickase strategy, two sgRNAs targeting adjacent sites guide two nCas9 molecules to introduce coordinated nicks on opposite DNA strands, resulting in a functional double‑strand break. This approach maintains editing efficiency comparable to wild‑type Cas9 but requires simultaneous recognition of two target sites, dramatically reducing tolerance for off‑target activity and significantly improving editing specificity.

Because of these advantages, nCas9(D10A)-sfGFP is widely used in high‑fidelity genome editing research and potential therapeutic applications.