CTS Cas9 Proteins

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).

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.
 

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.