32121 SpCas9 D10A Nickase
32121 SpCas9 D10A Nickase
-
Specifications：
- 100 pmoL
- 1,000 pmoL
Article number：
32121
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 D10 of SpCas9 has been mutated to A, rendering its RuvC domain inactive. As a result, SpCas9 D10A Nickase only has cleaving activity in the HNH domain and cleaves only the TS chain of the target dsDNA, creating a single-strand nick.
Product Components
| Components |
32121-01 (100 pmol) |
32121-03 (1,000 pmol) |
|
|
100 pmol |
1,000 pmol |
|
|
1 mL |
1 mL |
SpCas9 D10A Nickase (10 μM) a
10 × TOLO Buffer 3a.The concentration of SpCas9 D10A Nickase is 10 μM (10 pmol/μL). Therefore, for a 100 pmol, the total volume would be 10 μL, and for a1,000 pmol, the total volume would be 100 μL.
Storage Conditions
Store at -20℃; transport at ≤0℃.
Experiment Procedure
Notes
1. To prevent RNase contamination, please maintain a clean and tidy workspace. When handling, wear cleangloves and masks. All consumables such as pipette tips and centrifuge tubes should be RNase-free.
2. Since RNA (sgRNA or crRNA:tracrRNA) needs to be added to the reaction, the entire system, including the SpCas9 H840A Nickase enzyme, must be free from any RNase activity. This product has undergone strictquality control and is free from any nucleic acid enzyme contamination.
3. SpCas9 D10A Nickase is heat-sensitive and prone to inactivation. The entire reaction system should be prepared on ice, and after use, immediately store it at -20℃.
4. Complex formation between SpCas9 D10A Nickase and DNA/sgRNA can affect electrophoresis results. High-temperature inactivation at 85℃ is necessary before electrophoresis analysis.
5. This product is for research use only.
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).
References
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