Product Support
      Back to home
      1. Resource Center
      2. Product Support

      CRISPR Cas9 Nuclease: FAQs

      Frequently Asked Questions

      1. How does CRISPR Cas9 work?
        1. In the CRISPR Cas9 system, the Cas9 nuclease, guided by sgRNA (single guide RNA), directly performs targeted double-strand cleavage 3bp upstream of the DNA containing the protospacer adjacent motif (PAM) (i.e. NGG) to form site-specific DNA double-strand breaks (DSBs). These DSBs are repaired by inherent cellular repair mechanisms, such as non-homologous end joining (NHEJ) to achieve random insertion or deletion of bases, or homologous recombination repair (HR) to repair the cut site.
      2. What are the main differences between research-grade Cas9 nuclease and GMP-grade Cas9 nuclease?
        1. Production Environment: GMP-Grade Cas9 is produced in a high-standard GMP-certified manufacturing facility. Research-grade Cas9 is produced in a standard manufacturing facility.
        2. Quality Control: GMP-Grade quality control testing is more comprehensive than research-grade. Our GMP-Grade Cas9 undergoes 14 separate quality control tests before release.
        3. Documentation Support: GMP-Grade Cas9 includes a customizable documentation package including Datasheet, TSE/BSE Statment, COA, COO, MSDS, DMF, Melamine Statement, and Nitrosamine Statement. Batch production records and batch inspection records can also be provided. 
      3. What methods are used to detect the activity of KACTUS Cas9?
        1. We use two types of activity assays to analyze activity: in vitro cleavage activity and ex vivo knockout efficiency. Our in vitro cleavage activity assay assesses the cleavage activity of Cas9 by detecting the total mass ratio of two cleaved fragments formed and is a quality control release assay. Our ex vivo gene knockout efficiency was verified in 293T cells, Jurkat cells, and T cells. However, we do not verify the gene knockout efficiency of every Cas9 batch. Example activity data can be found on this page.
      4. What is the knockout efficiency of Kactus Cas9?
        1. We have analyzed gene knockout efficiency of KACTUS Cas9 enzyme versus leading suppliers in 293T, Jurkat, and primary T cells. Our GMP Cas9 had a very high gene knockout efficiency, and is also on par with leading suppliers. Howerver, the knockout efficiency depends on the cell type, target sequence, molar ratio of Cas9 to sgRNA, concentration of Cas9 to sgRNA, electroporator type, electroporation parameters, and other factors. We highly recommend testing out the enzyme in various conditions based on your project. Please reach out to use at sales@kactusbio.us to request a test sample. 
      5. What is the recommended quantity of Cas9 used?
        1. The amount of Cas9 used is related to the size of the electroporation system, type of electroporator, electroporation conditions, cell type, sgRNA targeting site, etc. For example, for 2×105 cells to 1×106 cells, we recommend trying 1-4μM (in a 25μL electrotransfer system) and do a gradient mapping of the dosage.
      6. For KACTUS Cas9, what is the molar concentration converted from 10mg/mL?
        1. molar concentration (M) = mass concentration (C)relative molecular mass (Mr) = 10mg/mL163000 g/mol = 61.35µM
      7. If I want to dilute Cas9, what buffer can I use for dilution?
        1. Our Cas9 concentration is 10mg/mL. Dilution is not recommended for use. If you have particular needs, we recommend using storage buffer without glycerol for dilution. If you need to store the Cas9 for a long time after dilution, we recommend using storage buffer for dilution. The storage buffer is 30mM Tris, pH 7.4, 300mM NaCl, 50% glycerol, 0.1mM EDTA.
      8. Are there any patent issues involved in the commercial production of Cas9?
        1. KACTUS Cas9 is wild-type spCas9, which is recombinantly expressed in E.coli. We have not used the modified sequences from other researchers for commercial production, so there are no patent issues. In addition, we have also performed FTO (Freedom To Operate) analysis, and our results show that our Cas9 has no risk of infringement and therefore has no patent restrictions. Generally CRISPR Cas9 patent refers to the technology method, and not to the enzyme itself. Cell and gene therapy companies using the technology to modify cells may need to pay royalties before the product becomes available. Cas9, an enzyme used in gene editing technology, is not subject to these patent issues itself.
      9. What are the shipping and storage conditions of Kactus Cas9?
        1. CRISPR Cas9 is shipped with dry ice and stored at -20±5°C.
      10. Can the Cas9 (CRISPR Associated Protein 9) ELISA Kit be used to detect Cas9 residues from other companies?
        1. This is not recommended. Our Cas9 (CRISPR Associated Protein 9) ELISA Kit is designed for the residual detection of KACTUS Cas9, and is able to quantify KACTUS Cas9 accurately. Other suppliers of Cas9 may have differences in sequence and production processes which may skew test results.
      11. What are the possible reasons for an overall low OD value detected by Cas9 (CRISPR Associated Protein 9) ELISA Kit during operation?
        1. The reagent addition was not performed according to the protocol.
        2. Incubation time and temperature were not performed according to protocol.
        3. There could be a wavelength selection error. Please make sure you read the value of OD450.
        4. The kit was not stored as required. Please note that some reagents are stored at -20°C and others at 2-8°C. Check the reagents themselves or the associated documentation here for more information.