Figure 1. CTS TrueCut Cas9 achieved comparable cell viability and editing efficiency to RUO Cas9 in T cells. CTS TrueCut Cas9 Protein and TrueCut Cas9 Protein v2 (7.5 pmol) were each mixed with TrueGuide Synthetic sgRNA (7.5 pmol) to form two RNP complexes targeting seven loci on four cell therapy-related genes. Each RNP complex was used to transfect 5x105 T cells using the Neon Transfection System (10 μL kit). Cells were harvested after 72 hours of culture and all reactions were performed in triplicate. (A) Cell viability, measured and analyzed by flow cytometry, exceeded 90% and was similar for all seven loci. (B) Editing efficiency, measured by targeted amplicon-seq validation (TAV) using an Ion Torrent NGS system, was also similar for all target loci.
High-performance Cas9 proteins manufactured following relevant GMP principles
As you translate your genome editing research from basic research to clinical settings, high-quality cell therapy ancillary materials and documentation are critical. Our Gibco Cell Therapy Systems (CTS) Cas9 proteins have been designed for clinical research and applications that require Cas9 nuclease manufactured to stringent specifications.
Stringent standards, exceptional performance
Our CTS Cas9 proteins are manufactured in compliance with standards for Ancillary Materials for Cell, Gene, and Tissue-Based Products including USP <1043>, Ph.Eur. 5.2.12, and ISO 20399 following the principles of 21 CFR Part 820 in an FDA-registered manufacturing site.
- Traceability documentation—Including COA, COO, Drug Master Files, and/or Regulatory Support Files* (available on request, at no additional charge)
- Extensive safety testing—Includes sterility, endotoxin, mycoplasma, residual nucleic acids, and proteins
- Aseptic manufacturing—Entire process completed using single-use technology
- Sterile filling—Finished product is packaged in a Class A/B (Class 100 and ISO 5 equivalents) cleanroom
*Country-specific Regulatory Support File process will be initiated upon request and will require additional documentation time.
CTS TrueCut Cas9 Protein
GMP-manufactured wild-type Cas9 protein designed to deliver maximum CRISPR editing efficiency
Intended usage:
Clinical research and applications
CTS HiFi Cas9 Protein
GMP-manufactured high-fidelity Cas9 protein designed to deliver improved specificity while maintaining high efficiency
Intended usage:
Clinical research requiring minimal off-target effects
CTS TrueCut Cas9 Protein
CTS TrueCut Cas9 Protein is a GMP-manufactured wild-type Cas9 protein designed to deliver maximum CRISPR editing efficiency.
- 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
- Large pack sizes—choose 2.5 or 5.0 mg with a 10 mg/mL concentration
Figure 2. CTS Cas9 achieved high HDR-based knock-in efficiency at 10x electroporation scale. CTS TrueCut Cas9 Protein and TrueCut Cas9 Protein v2 (12.5 μg or 75 pmol) was mixed with TrueGuide Synthetic sgRNA (75 pmol) and ssODN donor (150 pmol) to form RNP-ssODN targeting four therapeutic genes. Each RNP was transfected into 5x106 T cells using the larger-scale Neon Transfection System (100 μL kit). Cells were harvested after 72 hours of culture and all reactions were performed in triplicate. (A) Cell viability, measured and analyzed by flow cytometry, exceeded 90% for all genes and loci tested. (B) 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, was similar between the two Cas9 for all target loci.
Figure 3. CTS Cas9 achieved higher knockout efficiency than the competition. CTS TrueCut Cas9 Protein and a competitor’s Cas9 was mixed with TrueGuide Synthetic sgRNA targeting alpha and beta T cell receptor gene (TRAC and TRBC) regions to create Cas9-RNP complexes. Each Cas9-RNP complex was used to transfect 5x105 T cells using the Neon Transfection System. All reactions were performed in triplicate. (A) Sample flow cytometry data for TCR; overall, CTS Cas9 achieved over 88.7% KO efficiency compared to 61.7% for Supplier A Cas9. (B) Measured by NGS-based TAV, CTS Cas9 achieved higher average KO efficiency compared to Supplier A Cas9 at various targets (**p< 0.01).
For more sample data, including lot-to-lot consistency and stability under varying temperatures, see our CTS TrueCut Cas9 Protein application notes:
CTS TrueCut Cas9 Protein
CTS HiFi Cas9 Protein
Regulatory bodies like the FDA recognize the safety concerns of off-target cleavage events when using CRISPR-Cas9, particularly for therapeutics applications. To address these safety risks, we have developed CTS HiFi Cas9 Protein, a GMP-manufactured high-fidelity Cas9 protein designed to significantly reduce off-target events while maintaining high editing efficiency.
- Significantly reduce off-target effects — >90% reduction in a broad range of cell types including primary immune cells
- Equivalent editing efficiency — achieve the same high editing efficiency as CTS TrueCut Cas9 Protein, our wild-type protein designed for superior on-target editing efficiency
- Improved knock-in efficiency — also offered in our CTS HiFi Cas9 and CTS Xenon Genome Editing Buffer Kit to offer 2X improved knock-in efficiency
Gibco CTS HiFi Cas9 Protein significantly reduce the occurrence of off-target effects in human primary T cells and iPSC. 3 gRNAs were co-transfected with wild-type CTS TrueCut Cas9 Protein, CTS HiFi Cas9 Protein, and Supplier A’s GMP HiFi Cas9 using the Neon Transfection System. Target-enriched GUIDE-Seq (TEG-Seq), an Ion Torrent-adapted NGS method, was used for off-target detection. The red dots are on-target events normalized to 100%. Blue dots are off-target events plotted against the corresponding off-target to on-target ratio, which represents the risk probability of each off-target event. (Top) Improved off-target profiles in human primary T cells with CTS HiFi Cas9, (Bottom) Reduction in off-target events with CTS HiFi Cas9 in human iPSCs.
Gibco CTS HiFi Cas9 demonstrated equivalent on-target activity to wild-type CTS TrueCut Cas9 Protein. 5 gRNAs were co-transfected with CTS HiFi Cas9 Protein and wild-type CTS TrueCut Cas9 Protein using the Neon Transfection System. All reactions were performed in triplicate.
(A) Over 95% knockout efficiency at most targets. Protein knockout efficiency measured and analyzed by flow cytometry (Attune NxT Flow Cytometer). (B) Over 75% editing efficiency at all targets. Editing efficiency measured by an NGS-based system (Ion Torrent GeneStudio S5).
Up to 3 folds improvement in HDR% when using CTS HiFi Cas9 Protein with CTS Xenon GE Buffer. CTS HiFi Cas9 was mixed with TrueGuide Synthetic sgRNA and single stranded oligo (ssODN) to form two RNP-ssODN complexes targeting TRAC and CD52 gene. Each RNP-ssODN complex was delivered to primary T cells using the Neon Transfection System in CTS Xenon Genome Editing Buffer or Neon R Buffer (used as a control). All reactions were performed in triplicate. 3-fold improvement in the HDR efficiency for TRAC-4 (from 24% to 71%) and a 2.8-fold improvement for CD52-5 (from 18% to 50%). No significant impact to overall editing efficiency or cell viability was observed.
Exceptional performance with CTS HiFi Cas9 Protein in a CAR-T cell therapy workflow at all scales. CTS HiFi Cas9 and CTS TrueCut Cas9 (wild-type) were co-transfected with TRAC-encoded Invitrogen TrueGuide Synthetic sgRNA and a donor dsDNA chimeric antigen receptor (CAR) construct into primary human T-cells using the Neon NxT Electroporation System and CTS Xenon Electroporation systems to represent a small research scale and a large manufacturing scale, respectively. CTS Xenon Genome Editing Buffer was used across all scale. Cell viability, knockout and knock-in efficiency were measured at 10 days post electroporation. All reactions were performed in triplicate for each cell donor and two cell donors per each scale were performed. (A) Equivalent KO & KI performance to CTS TrueCut Cas9 (wild type) on the small research scale (B) Comparable performance on the large manufacturing scale.
CTS HiFi Cas9 Protein
Streamline your CRISPR off-target analysis
Our trusted, experienced team of scientists can help you monitor and detect off-target events in your gene editing experiment.
Frequently asked questions (FAQs) about CTS Cas9 proteins
Both products are the same Cas9 protein, but CTS Cas9 proteins are manufactured to meet the standards for Ancillary Materials for Cell, Gene, and Tissue-Based Products. This adds the benefits of traceability documentation, extensive safety testing, aseptic manufacturing, sterile filtration, high concentration, and large pack sizes as described above.
Please visitCell Therapy Systems Support for instructions.
Our Cas9 proteins are stored in a storage buffer solution composed of 10 mM Tris pH 8.0 (4°C), 100 mM NaCl, 200 mM Na2SO4, 50% glycerol.
Thermo Fisher Scientific’s Limited Use Label Licenses (LULLs)† associated with our Cas9 products address the use of CTS Cas9 proteins 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. For activities beyond this scope, it is the customer’s responsibility to obtain any required third-party rights.
† To view LULLs, follow the links to the respective product pages in the Order CTS Cas9 protein section below.
CTS Cas9 proteins release specifications
| Parameter | Specification |
|---|---|
| Concentration | 9.0-11.0 mg/mL |
| Activity Assay, in vitro | >90% in vitro of uncut reference DNA converted to cleavage products |
| Endotoxin | <10.0 EU/mg |
| Sterility | No growth |
| Purity by RP-HPLC | ≥95% |
| Aggregates | ≤5% |
| Identity, RP-HPLC | Conforms |
| pH | 7.0–7.8 |
| Residual DNase | <LOQ* |
| Residual RNase | <LOQ* |
| Residual host cell protein | <100.0 ng/mg |
| Residual host cell DNA | <LOQ* |
| Mycoplasma | <LOD** |
| *LOQ = limit of quantification; **LOD = limit of detection | |
CRISPR-Cas9 is driving a new generation of cell engineering for more specific cell-based therapies, such as CAR T cell therapy. Discover this evolution and key considerations for selecting Cas9 reagents for clinical applications.
For Research Use or Non-Commercial Manufacturing of Cell-Based Products for Clinical Research. CAUTION: Not intended for direct administration into humans or animals.