dCas9 Protein


Product Introduction

dCas9 (nuclease-dead Cas9) is an important variant derived from the CRISPR-Cas9 gene editing system through site-directed mutagenesis. This protein incorporates two key point mutations, D10A and N863A, which target the catalytic centers of the RuvC and HNH nuclease domains, respectively, thereby completely abolishing its endonuclease activity for cleaving double-stranded DNA. However, this modification does not affect its gRNA-guided DNA targeting ability. dCas9 retains the capacity to be precisely navigated by a guide RNA (gRNA) to specifically recognize and bind to particular DNA sequences within the genome; it simply ceases to perform the cleavage function of wild-type SpCas9 upon reaching its target location.

This characteristic of dCas9 establishes it as an ideal platform for gene regulation and functional studies. dCas9 can be utilized for dynamic imaging of specific genomic regions in living cells. When fused with epigenetic modifiers, it can precisely rewrite local epigenetic marks. These applications render dCas9 an indispensable tool in basic research, synthetic biology, and potential gene therapies, achieving a functional expansion from gene editing to gene regulation.

Product Specifications

Parameter

Specification

Source

Recombinant expression in E. coli

Molecular Weight

~161 kDa

Concentration

20 μM

PAM Sequence

5'-NGG-3'

Mutation Sites

D10A + N863A

Purity

≥95% (SDS-PAGE)

Endotoxin

<1 EU/μg

Storage Buffer

50 mM Tris-HCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol

10× Reaction Buffer

50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl₂, 100 μg/ml BSA, pH 7.9

Storage Conditions

Long-term storage at -80°C; short-term storage at -20°C

Product Specifications

Specifications

Catalog Number

Concentration

Volume

100 pmol

GR101701

20 μM

5 μL

500 pmol

GR101702

20 μM

25 μL

2500 pmol

GR101703

20 μM

125 μL

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.

Biosensing and Nucleic Acid Detection:

Immobilizing dCas9 on test strips or electrodes, coupled with a fluorophore. When the gRNA recognizes a viral RNA or a drug-resistance gene, dCas9 binds to the target and generates an optical or electrical signal.

Artificial DNA Looping and Chromatin Conformation Regulation:

Utilizing dCas9 in pairs, each binding to two distant DNA loci, and then bringing these loci together through protein dimerization. This is used to study the interaction between distant enhancers and promoters, or to artificially alter chromatin topology.

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.


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