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32120 SpCas9 H840A Nickase

  • 32120 SpCas9 H840A Nickase.png
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32120 SpCas9 H840A Nickase

  • Specifications:
    • 100 pmoL
    • 1,000 pmoL

Article number:

32120


Price:Contact our distributors


隐藏域元素占位

Product overview

Description

SpCas9 is derived from the S. pyogenes strain and is an RNA-guided DNA endonuclease that depends on crRNA and tracrRNA (or a fusion sgRNA) for guidance. In the presence of a PAM (NGG) sequence in the target dsDNA, it specifically cleaves the target DNA, creating a double-strand break and generating blunt ends. PAM is essential for both the recognition and cleavage by Cas9, with the cleavage site located within the target sequence, three bases away from the PAM region. The HNH and RuvC domains of SpCas9 enzyme are responsible for cleaving the target dsDNA that pairs with the sgRNA (target sequence or TS chain) and the non-paired strand (non-target sequence or NTS chain), respectively. In this product, the H840 of SpCas9 has been mutated to A, rendering its HNH domain inactive. As a result, SpCas9 H840A Nickase only has cleaving activity in the RuvC domain and cleaves only the NTS chain of the target dsDNA, creating a single-strand nick.

 

Product Components

Components

32120-01 (100 pmol)

32120-03 (1,000 pmol)

SpCas9 H840A Nickase (10 μM) a

100 pmol

1,000 pmol

10 × TOLO Buffer 3

1 mL

1 mL

a. The concentration of SpCas9 H840A Nickase is 10 μM (10 pmol/μL). Therefore, for a 100 pmol, the total volume would be 10 μL, and for a 1,000 pmol, the total volume would be 100 μL.

 

Storage Conditions

Store at -20℃; transport at ≤0℃.

 

 

Experimental Results

 

Fig1. nicking experiment.

 

 

Experiment Procedure

1.Supercoiled plasmid nicking experiment:

Components

Volume 

Final concentration 

10 × Tolo Buffer 3

2 μL 1 ×

10 μM SpCas9 H840A Nickase

0.04~0.05 μL 20~250 nM

10 μM sgRNA

0.08~1 μL 40~500 nM

1 μM Supercoiled DNA

0.2 μL 10 nM

Nuclease-free water

Up to 20 μL  

◆ If nucleic acid electrophoresis is used to analyze nicking products, for a 20 μL nicking system, it is recommended to use Supercoiled DNA in the range of 100 ng to 500 ng. The amounts of Cas enzyme and sgRNA required for different lengths of Supercoiled DNA should be calculated based on the molar quantity of Supercoiled DNA. It is recommended to maintain a molar ratio of Cas enzyme: sgRNA: Supercoiled DNA at 5:10:1 to ensure as complete nicking of Supercoiled DNA as possible.

FAQ

Q1: What is the difference between nCas9 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), together generating double-strand DNA breaks (DSBs) and producing blunt ends. However, nCas9, due to mutations like H840A or D10A in the amino acid sequence, inactivates either the HNH or RuvC domain, allowing it to cleave only one strand of the target dsDNA, creating a single-strand cut. Specifically, SpCas9 H840A Nickase can only cleave the non-target strand (NTS) of the target dsDNA that doesn't pair with the sgRNA, while SpCas9 D10A Nickase can only cleave the target strand (TS) that pairs with the sgRNA. Additionally, dCas9, with mutations in both H840A and D10A, can bind to the target dsDNA but cannot cleave either strand.

 

Q2: How to design the sgRNA for nCas9?

A2: The sgRNA for nCas9 can be designed based on the following sequence:
5′–NNNNNNNNNNNNNNNNNNNNUUUCGUGGCUGAGCCACGGUGAAAAAGUUCAACUAUUGCCUGAUCGGAAUAAAAUUGAACGAUAAAGAUCGAGAUUUUG–3′, (The underlined portion represents the spacer region that complements the specific target sequence).

Product Instruction

#32120 SpCas9 H840A Nickase

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References

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