High-performance Cas9 protein manufactured following relevant GMP principles

CTS TrueCut Cas9 Protein


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) TrueCut Cas9 Protein formulation is designed for clinical research and applications that require wild-type Cas9 nuclease manufactured to stringent specifications.

  • 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


Stringent standards, exceptional performance

CTS TrueCut Cas9 Protein is 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 -1, -2, and -3, 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)
  • 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.

Data: CTS TrueCut Cas9 Protein achieves high knockout efficiency in primary T cells

Two bar graphs demonstrating similar cell viability and cleavage activity levels in T cells transfected with either CTS or RUO Cas9 proteins

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.

Data: CTS TrueCut Cas9 Protein achieves high knock-in efficiency in primary T cells

Two bar graphs demonstrating that T cells transfected with Gibco CTS TrueCut Cas9 exhibited high levels of cell viability and knock-in efficiency for experiments targeting different loci

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.

Data: CTS TrueCut Cas9 Protein knockout efficiency surpasses the competition in primary T cells

A flow cytometry chart shown above a bar graph, both demonstrating that Gibco CTS TrueCut Cas9 Protein achieved greater TCR knockout compared to competitor’s GMP Cas9

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 release specifications

ParameterSpecification
Concentration9.0-11.0 mg/mL
Activity Assay, in vitro>90% in vitro of uncut reference DNA converted to cleavage products
Endotoxin<10.0 EU/mg
SterilityNo growth
Purity by RP-HPLC≥95%
Purity by SDS-PAGE≥95%
Aggregates≤5%
Identity, RP-HPLCConforms
Identity, SDS-PAGEConforms
pH7.0–7.8
Residual DNase<LOQ*
Residual RNase<LOQ*
Residual host cell protein<LOQ*
Residual host cell DNA<LOQ*
Mycoplasma<LOD**
*LOQ = limit of quantification; **LOD = limit of detection


Frequently asked questions (FAQs) about CTS TrueCut Cas9 Protein

Both products are the same Cas9 protein, but CTS TrueCut Cas9 Protein is 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 filling, high concentration, and large pack sizes as described above.
 

Our Cas9 protein is 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 TrueCut Cas9 Protein 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.
 



Order CTS TrueCut Cas9 Protein

Order CTS TrueCut Cas9 Protein here or use our TrueDesign Genome Editor to design and order materials for your entire edit.

For  Research Use or Non-Commercial Manufacturing of Cell-Based Products for Clinical Research. CAUTION: Not intended for direct administration into humans or animals.