The Cas9 Nuclease (SpCas9) produced by our company—also known as CRISPR-associated endonuclease Cas9 or Csn1—is an endonuclease derived from Streptococcus pyogenes. It is expressed and purified through our independently developed technology platform, and can sequence-specifically cleave double-stranded DNA under the guidance of gRNA.This product is applicable for multiple uses, including in vitro screening of high-efficiency guide RNA (gRNA) sequences, gRNA-directed cleavage of specific DNA sequences, and linearization of double-stranded circular DNA containing target sequences.CRISPR/Cas9 is a breakthrough genome editing technology with simple operation and wide applications. CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system in prokaryotes. It uses the RNA-guided DNA nuclease Cas9 to silence foreign phage or viral nucleic acids. Subsequently, it has evolved into an increasingly mature gene editing technology widely used in both prokaryotes and eukaryotes.The technology enables site-specific cleavage of target sequences in the genomic DNA of prokaryotes and eukaryotes via Cas9 under gRNA guidance. Frameshift mutations are then generated by altering or inserting sequences at the cleavage site through error-prone repair or homologous recombination, thereby achieving gene knockout through gene editing. Among these, gRNA ensures the specificity of the recognition site.With the development of CRISPR technology, it now supports multiple mutation types beyond gene knockout, such as gene point mutations and insertion mutations. It is particularly useful in clinical applications, including the repair of harmful mutations. Additionally, by constructing dCas9—a Cas9 mutant without endonuclease activity—transcriptional activation or inhibition of sgRNA-targeted genes can be achieved through direct fusion expression with dCas9 or indirect recruitment of transcriptional activators or repressors.
Purity: Free of exonuclease, non-gRNA-dependent endonuclease, and RNase.
| C744422 | Component | 50pmol | 5*50pmol | 20*50pmol | Storage | | C744422A | Cas9 Nuclease (1μM) | 50µl | 5*50µl | 20*50µl | -20℃. Avoid freeze/thaw cycle. | | C744422B | Cas9 Reaction Buffer (10X) | 0.2ml | 5*0.2ml | 20*0.2ml | -20℃. Avoid freeze/thaw cycle. |
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Product Applications
In vitro screening of high-efficiency gRNA sequences, gRNA-guided cleavage of specific double-stranded DNA, selective linearization of double-stranded DNA containing specific sequences, etc.
Product Advantages
Cas9 Nuclease can form a stable ribonucleoprotein (RNP) complex with guide RNA (gRNA), which enters the nucleus to achieve efficient genomic DNA cleavage. It exhibits stable and high editing efficiency in various cell lines, including standard cell lines, immune cells, primary cells and stem cells.
Usage Instructions
1. Dissolve and mix all solutions required for in vitro digestion reactions. Place Cas9 Nuclease, gRNA and substrate DNA on ice. Dilute gRNA to 300nM and substrate DNA to 30nM using nuclease-free water.
2. Prepare the reaction system according to the following table (taking 30μl system as an example):
| Reagent | Volume | | Nuclease-free Water | 20µl | | Cas9 Reaction Buffer (10X) | 3µl | | gRNA (300nM) | 3µl | | Cas9 Nuclease (1μM) | 1µl | | Total Reaction Volume | 27µl |
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3. Gently mix by pipetting up and down or slight vortexing. Centrifuge briefly at room temperature to collect the liquid at the bottom of the tube. Pre-incubate at 25℃ for 10 minutes.
4. Add 3μl of 30nM substrate target DNA (final volume 30μl). Mix gently (by pipetting up and down or light vortexing at the lowest speed), then centrifuge to pellet the liquid. Incubate at 37℃ for 15 minutes; the reaction time can be appropriately extended to 30-120 minutes depending on actual conditions.
5. Add 1μl of Proteinase K to each sample, mix gently, and incubate at room temperature for 10 minutes.
6. Add 6μl of 6X DNA Loading Buffer to each reaction system, then perform electrophoresis analysis using an appropriate concentration of agarose gel. If electrophoresis is not performed immediately, the sample can be stored at -20℃ for later use.
Frequently Asked Questions
1. Why is the target DNA cleavage incomplete?
a. It may be caused by an inappropriate molar ratio of Cas9 Nuclease, sgRNA, and target DNA. A molar ratio of at least 10:10:1 for Cas9 Nuclease:sgRNA:target DNA is recommended. The reaction can also be made more complete by appropriately extending the reaction time.
b. It may be related to the sgRNA sequence. A more suitable sgRNA sequence can be selected based on the target DNA, as the efficiency of different sgRNAs varies significantly.
c. It may be caused by sgRNA degradation. The integrity of sgRNA can be verified by gel electrophoresis.
Storage Conditions
Store at -20℃ and transport at ≤0℃.
Precautions
(1) The use of this product involves operations with gRNA and DNA, so strict RNase-free and DNase-free practices must be followed. All self-prepared reagents and consumables should also be Nuclease-free. If nuclease contamination is possible, treat with 0.01% DEPC overnight, followed by autoclaving before use. It is recommended to wear a disposable mask during operation.(2) For the removal of nucleases in the operating environment, Aladdin's RNase and DNase Away (R749971) is recommended to eliminate nucleases on surfaces such as laboratory benches, instruments, and other contact surfaces. Adding RNase Inhibitor to the reaction system is recommended to protect RNA from degradation.
(3) This product is for scientific research use only by professional personnel. It shall not be used for clinical diagnosis or treatment, food or pharmaceuticals, nor stored in ordinary residences.(4) For your safety and health, wear a lab coat and disposable gloves during operation.
