32102 Sp-dCas9 Nuclease
32102 Sp-dCas9 Nuclease
-
Specifications：
- 100 pmoL
- 1,000 pmoL
Article number：
32102
Price：Contact our distributors
隐藏域元素占位
Product overview
Description
SpCas9 is a DNA endonuclease guided by crRNA and tracrRNA (or sgRNA alone), which is derived from S. pyogenes. SpCas9 specifically recognizes and cleaves dsDNA target in a PAM (NGG)-dependent manner. The cleavage site of Cas9 in the target sequence is 3 bps away from the PAM site. The HNH and RuvC domains of SpCas9 are responsible for cleaving the target and non-target strands of dsDNA, respectively. In Sp-dCas9, both H840 and D10 are mutated to A, so that its HNH and RuvC domain are inactivated and Sp-dCas9 has only the target DNA binding activity, but not the cleaving activity.
Product Components
| Components |
32102-01（100 pmol） |
32102-03（1,000 pmol） |
|
|
100 pmol | 1,000 pmol |
|
|
1 mL | 1 mL |
Sp-dCas9 Nuclease (10 μM) a
10 × TOLO Buffer 3a.The concentration of Sp-dCas9 Nuclease is 10 μM (10 pmol/μL);
Storage Conditions
Store at -20℃; transport at ≤0℃.
Experimental Results
In order to verify that Sp-dCas9 could specifically bind to target DNA under the guidance of sgRNA, we introduced a FAM labeling to the 5′ end of the 59-bp dsDNA target. The reaction system included dsDNA target, sgRNA, and Sp-dCas9 Nuclease, and was incubated at 37°C for 30 min. The reaction product was analyzed by 2% agarose gel under a low temperature, and was then analyzed by electrophoresis (100 V, 60 min), followed by detection with the FAM fluorescence channel on GE ImageQuant-LAS4000. The experimental results showed that the electrophoresis migration rate was slower in the lane with Sp-dCas9 + sgRNA + dsDNA target-FAM, indicating that Sp-dCas9 specifically recognized and bound dsDNA target under the guidance of sgRNA. Alternative to agarose gel analysis, the binding of Sp-dCas9 to target DNA can also be detected by non-denaturing polyacrylamide gel electrophoresis (Native-PAGE).
Fig1. Sp-dCas9 specifically recognizes and binds target dsDNA under the guidance of sgRNA.
Experiment Procedure
1. The binding experiment of Sp-dCas9 and target DNA
|
Components |
Volume |
Final concentration |
|
10 × TOLO Buffer 3 |
2 μL | 1 × |
|
10 μM Sp-dCas9 Nuclease |
1 μL | 500 nM |
|
10 μM sgRNA |
1.5 μL | 750 nM |
|
1 μM Substrate DNA |
3 μL | 150 nM |
|
Nuclease-free water |
Up to 20 μL |
◆ Incubate at 37℃ for 30 min. The experimental results can be detected by non-denaturing nucleic acid electrophoresis.
FAQ
Q1: What are the differences between dCas9 and Cas9?
A1: Cas9 can recognize and cleave dsDNA targets, and its mechanism involves two structural domains, HNH and RuvC. HNH cleaves the target strand (TS), while RuvC cleaves the non-target strand (NTS), resulting in double-strand DNA breaks (DSBs) and creating blunt ends in the target DNA. However, dCas9, due to amino acid mutations (H840A and D10A), renders the HNH and RuvC domains inactive. As a result, dCas9 can bind to the target dsDNA but cannot cleave it. Additionally, there is also nCas9, where a D10A mutation inactivates the RuvC domain, allowing nCas9 to bind to dsDNA and cleave only one strand, creating a nick.
Q2: How to design the sgRNA for Sp-dCas9?
A2: The sgRNA for Sp-dCas9 can be designed based on the following sequence:
5′–NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG–3′, (The underlined portion represents the spacer region that complements the specific target sequence).
References
