Cas9 Protein for clinical research applications
As you move your genome editing research from basic research to clinical settings, high-quality cell therapy ancillary materials and documentation are critical. We now offer our Gibco CTS TrueCut Cas9 Protein for clinical research that requires next generation Cas9 nuclease that is manufactured with stringent specifications.
Key Benefits of Gibco CTS TrueCut Cas9 Protein
- Consistent high editing efficiency—in all tested cell lines, including >90% editing in primary T cells
- Higher stringency manufacturing—Cas9 protein manufactured in compliance with the principles of GMP and to meet the standards for Ancillary Materials for Cell, Gene and Tissue-Based Products including USP <1043>, Ph.Eur. 5.2.12, and ISO 20399 -1, -2, -3. This includes the following added benefits:
- Traceability documentation—including COO, COA, Drug Master Files and/or Regulatory Support Files
- Extensive safety & release testing—including sterility, endotoxin, mycoplasma, residual host nucleic acids, and proteins
- Aseptic manufacturing—entire manufacturing process completed using single-use technology
- Sterile filtration—finished product is vialed in a Class A/B (Class 100 and ISO 5 equivalents) Cleanroom
- Large pack sizes—2.5 and 5.0 mg with a 10 mg/mL concentration
Figure 1. Consistent cell viability and editing efficiency between Gibco CTS TrueCut Cas9 and Invitrogen TrueCut Cas9 v2 in human primary T-cells. Gibco CTS TrueCut Cas9 (Cat. No. A45220) and Invitrogen TrueCut Cas9 Protein v2 (Cat. No. A36499) were each complexed with TrueGuide™ Synthetic sgRNA (Cat. No. A35534). Each RNP complex was used to transfect 5x105 T cells using the Neon Transfection System (Cat.No. MPK5000). Cells were harvested after 72 hours of culture. (A) Cell viability was measured and analyzed by Attune NXT flow cytometer. (B) Summary of NGS-based analysis of cleavage efficiency as measured by targeted amplicon-seq validation (TAV) using Ion Torrent™ NGS system for all target loci. All reactions were performed in triplicate.
Figure 2. Gibco CTS TrueCut Cas9 achieved high HDR-based knock-in efficiency at 10X electroporation scale. Gibco CTS TrueCut Cas9 (Cat. No. A45220) and Invitrogen TrueCut Cas9 Protein v2 (Cat. No. A36499) were complexed with TrueGuide™ Synthetic sgRNA (Cat. No. A35534) and ssODN donor to form RNP-ssODN. Each RNP complex was used to transfect 50 x 105 T cells using the larger-scale Neon Transfection System (Cat. No. MPK10025). Cells were harvested after 72 hours of culture. (A) Cell viability was measured and analyzed by Attune NXT flow cytometer. (B) Summary of editing efficiency calculated as percentage of donor integration through HDR and indel as measured by targeted amplicon-seq validation (TAV) using an Ion Torrent NGS system. All reactions were performed in triplicate.
Figure 3. Achieve higher knockout efficiency with CTS Cas9 compared to the competition. Each Cas9 and Invitrogen™ TrueGuide™ Synthetic sgRNA targeting alpha and beta T cell receptor gene (TRAC and TRBC) regions were mixed to create Cas9-RNP complexes. Each Cas9-RNP complex was used to transfect 5x105 T cells using the Neon Transfection System (Cat. No. MPK5000). (A) An example of flow cytometry data for TCR KO efficiency. CTS Cas9 achieved over 88.7% KO efficiency compared to Supplier A Cas9, which achieved 61.7% KO efficiency. (B) Average KO efficiency from NGS-based TAV. CTS Cas9 achieved higher average KO efficiency compared to Supplier A Cas9 at various targets. All reactions were performed in triplicate (** P< 0.01).
Figure 4. Achieve consistent performance across multiple lots with CTS Cas9 in primary T cells. CTS Cas9 proteins from three manufacturing lots were serially diluted. Different amounts of CTS Cas9 were mixed with (A) TRAC-4 or (B) CD52-6 TrueGuide Synthetic sgRNA and transfected into T cells using the Neon Transfection System (10 μL kit).
Figure 5. CTS TrueCut Cas9 remained stable after five freeze-thaw cycles as measured using an in vitro cleavage assay. (A) CTS Cas9 was serially diluted to the linear range of dose and the cleavage activity was measured. Different amounts (8 ng, 16 ng, and 32 ng) of CTS Cas9 were incubated with excessive gRNA (40 ng) targeting HPRT gene and a plasmid (300 ng) containing HPRT sequence for 10 minutes at 37°C. (B) The cleavage reactions containing uncut and cut plasmids were dissolved in agarose gel and quantitated using an Invitrogen™ iBright™ Imaging System. Reactions were performed in triplicate.
Figure 6. CTS Cas9–RNP complex maintained cleavage efficiency at room temperature for up to 16 hours. CTS Cas9 was serially diluted to the linear range of dosage, and the cleavage activity was measured. Different amounts of CTS Cas9 were mixed with an excess of gRNA targeting the HPRT gene, and incubated for different time points at room temperature. (A) The cleavage reactions containing uncut and cut plasmids were resolved on an agarose gel and quantitated using an iBright Imaging System. (B) The cleavage activity plotted as a bar graph. Reactions were performed in triplicate.
|Activity Assay, in vitro||>90% in vitro of uncut reference DNA converted to cleavage products|
|Endotoxin Testing||<10.0 EU/mg|
|Residual Host Cell Protein||<LOQ|
|Residual Host Cell DNA||<LOQ, qPCR|
|Mycoplasma||Negative by qPCR|
1. How is Gibco CTS TrueCut Cas9 Protein different from TrueCut Cas9 Protein v2?
Gibco CTS TrueCut Cas9 Protein is manufactured to meet the standards for Ancillary Materials for Cell, Gene, and Tissue-Based Products. Extensive safety testing is performed. TrueCut Cas9 Protein v2 is a research grade wild type Cas9 enzyme with added NLS to improve delivery to the nucleus.
2. What is the storage buffer composition for the Gibco CTS TrueCut Cas9?
Our Cas9 protein is stored in a storage buffer solution. The storage buffer composition is: 10 mM Tris pH 8.0 (4°C), 100 mM NaCl, 200 mM Na2SO4, 50% glycerol
3. How do I request traceability documentation for the Gibco CTS TrueCut Cas9?
For instructions on how to request traceability documentation, please visit Cell Therapy Systems Support webpage.
4. CRISPR has a lot of patents. Does Thermo Fisher Scientific have clinical rights?
Thermo Fisher Scientific’s Limited Use Label licenses associated with our Cas9 products address the use of TrueCut Cas9 Protein (CTS-Prototype) for discovery research, pre-clinical research, and clinical research activities that fall within safe harbor statutes of 35 U.S.C. §271(e)(1). The scope of activities that are covered by 35 U.S.C. §271(e)(1) needs to be determined by the customer. Anything beyond this scope is on the customer to obtain any required third-party rights.
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TrueGuide Synthetic Guide RNA
TrueCut Cas9 proteins must be combined with a CRISPR guide RNA (gRNA), to produce a functional, target-specific editing complex. Our proprietary gRNA design algorithms select gRNAs for maxium editing efficiency without compromising specificity.
For discovery and pre-clinical work, check out our TrueGuide Synthetic gRNAs, predesigned for highly efficient gene knockout, or visit the TrueDesign Genome Editor to design custom gRNAs and DNA donors for precise genome engineering.