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32102 Sp-dCas9 Nuclease

  • 32102 Sp-dCas9 Nuclease.png
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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) 

 Sp-dCas9 Nuclease (10 μM) a

100 pmol 1,000 pmol

 10 × TOLO Buffer 3

1 mL 1 mL

a.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).

Product Instruction

#32102 Sp-dCas9 Nuclease

pdf

490.0KB

References

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