Highly-active GMP-grade CRISPR Cas9 protein
Due to the ease of design and operation, the CRISPR-Cas system has become the most widely used gene editing technology today. At KACTUS, we have developed a highly-active, wild-type Cas9 nuclease based on our SAMS™ technology platform. Our Cas9 nuclease has been codon-optimized and process-optimized to reach the best yield and purity. The addition of the nuclear localization signal (NLS) makes it suitable for gene editing work in eukaryotic cells.
Our GMP-grade, recombinant CRISPR Cas9 protein meets the highest quality and performance standards for gene editing research and manufacturing. Our GMP-grade Cas9 enzyme is manufactured in compliance with cGMP standards and accompanied by comprehensive quality documentation for regulatory support, making it eligible for CGT process development and CMC manufacturing. Finally, with a well-established global supply chain, we are capable of stably supplying our Cas9 enzyme in bulk quantities worldwide.
Why choose our Cas9 protein?
GMP Production
Our GMP-grade Cas9 is manufactured in compliance with GMP standards, ensuring the highest levels of quality and safety. We offer both research-grade and GMP-grade versions to seamlessly transition from preclinical study to clinical/commercial production.
Analytical Method Validation
We validate all analytical methods according to pharmacopeia standards, ensuring consistent performance and reliability of our CRISPR Cas9 protein. This rigorous validation process guarantees that our product meets the most demanding quality benchmarks in the industry.
Batch Consistency
Our rigorous quality control ensures every batch of our CRISPR Cas9 protein is manufactured with consistent quality and activity, providing you with a reliable product that delivers solid, consistent results every time. This consistency is critical for reproducible research outcomes and regulatory submissions.
High editing activity
Our CRISPR Cas9 protein demonstrates high editing activity, both in vitro and in vivo, enabling precise and efficient genome editing. This ensures that your gene editing experiments are both effective and reliable, reducing the need for extensive optimization.
QC & Stability Testing
We conduct comprehensive sterility and stability testing on our CRISPR Cas9 protein to ensure that it remains effective and safe over time. This confirms the protein’s longevity and reliability, even under varying storage and usage conditions. This documentation is also available to support regulatory filings.
Quality Documentation
We provide comprehensive quality documentation to support your IND and BLA filings, This includes animal-free origin statements (TSE/BSE), certificates of analysis, batch records, and other required documents to facilitate smooth regulatory submissions and approvals.
Product Performance Data
We've conducted extensive performance and analytical testing on our Cas9 protein to ensure high editing activity and quality. Our GMP-grade Cas9 protein demonstrates strong editing activity across various cell types, comparable to industry standards. We showcase successful gene knockouts in diverse cell lines, including 293T, Jurkat, and primary T cells, with editing efficacies greater than 85%. Our team has also conducted comprehensive stability testing on the Cas9 enzyme to underscore its reliability for use clinical and commercial applications.
High Editing Activity in Multiple Cell Types
(A) BCL11A Knockout in 293T Cells
(B) BCL11A Knockout in Jurkat Cells
(C) TRAC Knockout in Primary T Cells
Figure 1. Gene knockout in 293T (A), Jurkat (B), and T cells (C) with different batches of KACTUS GMP-grade Cas9 protein. Results show greater than 85% editing efficacy across all three cell types, comparable to a leading supplier. Briefly, 75 pmol of Cas9 protein was electroported along with 225 pmol of sgRNA into various cells using the Lonza 4D Nucleofector system. The cells were cultured for three days followed by TIDE analysis. Gene knockouts: (A) knockout BCL11A in 293T cells; (B) knockout TRAC in Primary T cells; (C) knockout BCL11A in Jurkat cells.
Comparable activity to leading suppliers
Figure 2. Comparison of gene knockout efficiency using Cas9 nucleases from KACTUS and two leading suppliers in UCAR-T cells. Gene knockout efficiency was assessed at 72 hours and 120 hours post-electroporation. Results indicate that KACTUS Cas9 nuclease performs comparably to leading suppliers, effectively knocking out genes related to immune rejection reactions on donor T cells.
Figure 3. Gene knockout efficiency of BCL11A in hematopoietic stem cells (HSCs) for the treatment of β-thalassemia using Cas9 nucleases from KACTUS and two leading suppliers. Results demonstrate that KACTUS Cas9 nuclease achieves gene knockout efficiency comparable to that of the leading suppliers, validating its effectiveness for therapeutic applications.
Batch consistency and long-term stability
Our rigorous quality control ensures every batch of our CRISPR Cas9 protein is manufactured with consistent quality and activity, providing you with a reliable product that delivers solid, consistent results every time. This consistency is critical for reproducible research outcomes and regulatory submissions.
Figure 4. Consistent purity and activity performance was observed across three manufacturing batches of KACTUS GMP-grade Cas9. Three batches of KACTUS GMP-grade Cas9 were stored at -20°C for long-term stability testing. Purity and in vitro cleavage activity were analyzed at 3, 6, 9, 12, 15, 18 months, respectively. Results show our GMP-grade Cas9 has a good stability and consistent activity across time and different manufacturing batches.
Product Specifications & Quality
Product Specifications
Both our GMP-grade and research-grade Cas9 protein adhere to the following product specifications.
| Parameter | Specification |
|---|---|
| Express System | E. coli with CRISPR Cas9 gene of S. pyogenes |
| Concentration | 9.5-12.5 mg/mL |
| Form | Liquid |
| Storage Buffer | 30mM Tris-HCl, 0.3M NaCl, 50% Glycerol, 0.1 mM EDTA, pH 7.4 |
| Storage | -20±5°C for 18 months |
| Purity (Bis-Tris) | ≥ 95.0% |
| Purity (SEC-HPLC) | ≥ 95.0% |
| Activity | > 85.0% |
| Residual RNase | Negative |
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Quality Control Criteria
Each batch of our GMP-grade Cas9 Protein is tested for and meets the quality control criteria defined in the table below. Our research-grade Cas9 protein demonstrates similar performance but does not undergo lot-specific testing for all QC criteria.
| Assay | Acceptance Criteria | Appearance | Clear Liquid |
|---|---|
| Loading Amount | Not less than the amount of identification |
| Concentration | 9.5-11.5mg/mL |
| Identification | Corresponding to Reference Spectrum |
| Purity (RP-HPLC) | ≥ 95.0% |
| Purity (SEC-HPLC) | Monomer ≥ 95.0%, Aggregates ≤ 5.0% |
| Purity (NR-CE) | ≥ 85.0% |
| Purity (R-CE) | ≥ 90.0% |
| Bacterial Endotoxin | ≤ 10.0 EU/mg, (LOQ=0.1EU/mL) |
| Activity | ≥ 85.0% |
| Residual DNase | Negative (LOD=0.33U/μL) |
| Residual RNase | Negative (LOD=2.2×10⁻⁷U/μL) |
| Residual Host Cell Protein | ≤ 100.0 ng/mL (LOQ=3ng/mL) |
| Residual Host Cell DNA | ≤ 3.0 ng/mL (LOQ=0.03ng/mL) |
| Sterility | No growth |
| Residual Nickel Salt | ≤ 10.0 ppm (LOD=10.0ppm) |
| pH | 7.4±0.5 |
GMP-Grade Production
Our GMP-Grade Cas9 Protein is produced following cGMP standards, ensuring the traceability of raw materials, manufacturing process and QC release. This Cas9 protein is available in long-term bulk supply with batch-to-batch consistency to ensure suitability for industrial applications.
Documentation Package
Cas9 nuclease, GMP-grade comes with customizable regulatory documentation including Data Sheet, MSDS, COA, TSE/BSE statement, CoO. KACTUS has an integrative documentation system consisting of a digital quality management system, specification program, criteria support documents, and quality reports. Our product release process involves 15+ contaminant detection steps, 30+ quality tests, 4+ report reviews, and 4+ product release checkpoints. The documentation package is customizable based on relevant regulatory filings.
Quality Management System
→ ISO13485 Accreditation
→ Digital Manufacturing Execution System (MES)
→ Process and Analytical method validation
→ Batch-to-batch stability and consistency
→ Free from antibiotic residues and raw materials of animal origin
→ Comprehensive records for batch production
→ Pharmaceutical Class A & C Clean Room
→ Validated and maintained equipment
Universal Cas9 Protein ELISA kit
In ex vivo gene therapy, after cells are edited, the residual level of Cas9 protein must be measured before being reinfused into the patients. KACTUS has carefully developed a highly sensitive residual detection kit, the Cas9 (CRISPR Associated Protein 9) ELISA Kit. Our Cas9 ELISA Kit has also been validated with other leading commercial vendors and performs well as a universal Cas9 protein detection kit. The kit uses monoclonal antibodies in a sandwich ELISA and demonstrates high accuracy.
Figure 5. Example 8-point standard curve for Cas9 Protein ELISA kit.
Figure 6. Detection of various suppliers Cas9 protein using KACTUS Cas9 ELISA Kit. The Cas9 products were serially diluted 3-fold from 1000ng/mL and tested following the KACTUS Cas9 ELISA kit procedure. Results show that the KACTUS Cas9 ELISA kit can be used for the quantification of various vendors' Cas9 protein. The sensitivity for other vendors was 0.2-0.4ng/mL, which is close to the sensitivity for KACTUS Cas9 protein (0.125ng/mL).
FAQs
Product Specifications
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.
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.
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.
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.
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.
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.
Product Usage
The quantity 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.
molar concentration (M) = mass concentration (C) / relative molecular mass (Mr) = (10mg/mL) / (163000g/mol) = 61.35µM
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.
Manufacturing and selling the wildtype Cas9 protein itself does not present any patent issues. This means that companies can produce and distribute the wild-type version of Cas9 without infringing on patents. However, if a user wants to purchase the wild-type Cas9 for the purpose of commercial manufacturing, particularly if the Cas9 will be used in therapeutic applications, they must obtain a license from the IP owner who developed the specific therapeutic method that uses Cas9 for treatment.
In summary, while the Cas9 protein itself is not under patent restriction, the use of Cas9 in certain therapeutic contexts may be patented, and those patents need to be respected through proper licensing agreements.
- The reagent addition was not performed according to the protocol.
- Incubation time and temperature were not performed according to protocol.
- There could be a wavelength selection error. Please make sure you read the value of OD450.
- 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.
Ordering Information
You can order GMP-grade or research-grade Cas9 protein directly online via credit card or PO. Please send PO information to orders@kactusbio.us. To request a quote, please contact sales@kactusbio.us.
Research-grade Cas9 protein is available in 100UG / 1MG. GMP-Grade AccuBase™ is available in 3MG. Bulk sizes are available. Please contact sales@kactusbio.us.
CRISPR Cas9 is shipped with dry ice and stored at -20±5°C.
Cas9 protein, both the research-grade and GMP-grade, is an off-the-shelf product. The lead time is up to two weeks. However, some smaller sizes may ship out next day. For bulk orders, please contact sales@kactusbio.us to request a specific lead time.
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Product Brochure
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Research Poster
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GMP-Grade CRISPR Cas9 Protein