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Cat. No.: SNAP9-100 (for 100pmol)

Cat. No.: SNAP9-500 (for 500pmol)

Description

SNAP-dCas9 is a catalytically inactive Cas9 nuclease mutant (D10A/H840A) fused with a nuclear localization signal (NLS) and an O6-alkylguanine-DNA-alkyltransferase tag (SNAP-tag). It binds double-stranded DNA in a sequence-specific manner under the guidance of guide RNA (gRNA) yet lacks DNA cleavage activity.

Like wild-type Cas9 nuclease, dCas9 relies on gRNA to target desired DNA sequences. However, mutations within the RuvC and HNH domains abolish its endonuclease activity, leaving only gRNA-dependent DNA-binding capability.

The SNAP-tag is a genetically engineered variant of human O6-alkylguanine-DNA alkyltransferase (hAGT). It forms irreversible, exceptionally stable covalent bonds with O6-benzylguanine (BG) with high specificity. When BG is conjugated to a detectable reporter, the reporter is stably transferred onto the SNAP-tag upon this reaction. As an advanced tool for protein research, SNAP-tag features outstanding specificity, robust stability, and flexible, straightforward downstream labeling. With a molecular weight of approximately 20 kDa, it serves as an ideal protein fusion tag with multiple merits listed below:

• The reaction kinetics between SNAP-tag and benzylguanine (BG) derivatives are largely unaffected by the identity of the conjugated reporter moiety.
• SNAP-tag fusion rarely disturbs subcellular protein localization and imposes no restrictions on expression hosts.
• SNAP-tag substrates are chemically inert toward endogenous cellular proteins, eliminating non-specific labeling in live-cell applications.
• A wide range of SNAP-tag substrates exhibit cell membrane permeability, enabling labeling of intracellular proteins in living cells.
• Benzylguanine substrates conjugated with diverse reporters can covalently attach to SNAP-tag, allowing a single tagged protein to be labeled with multiple distinct probes with ease.

SNAP-dCas9 bears an N-terminal SNAP-tag and a C-terminal NLS. After transfection into cells, the NLS facilitates nuclear import of the SNAP-dCas9/gRNA ribonucleoprotein complex from the cytoplasm. Subsequently, gRNA directs SNAP-dCas9 to its target genomic DNA locus. The reactive cysteine residue on the N-terminal SNAP-tag reacts with the benzyl side chain of benzylguanine; guanine is cleaved off, and a stable thioether linkage covalently couples the BG-conjugated reporter to SNAP-dCas9. Compatible reporters include CY3, CY5, AF488, AF555, AF549, AF647, FITC, biotin and more, enabling direct visualization of specific genomic loci within cells (Figure 1).

SNAP-dCas9 can be delivered into cells via microinjection, electroporation, or liposome-mediated transfection. Notably, this DNA-free delivery system eliminates the risk of exogenous DNA integration into the host cellular genome.

Source

Produced via recombinant expression and purification in E. coli. The gene encoding the catalytically inactive Cas9 mutant (D10A/H840A) is derived from Streptococcus pyogenes.

Applications

Transcriptional regulation of target genes, genomic locus imaging, gene enrichment, and related research.

Purity

Protein purity ≥95% as determined by SDS-PAGE analysis; free of DNA exonuclease, DNA endonuclease, and RNase contaminants.

Storage