Cas9 Proteins

CRISPR gene editing with TrueCut Cas9 Protein v2 achieved robust editing efficiencies in human primary T cells with functional knockout up to 90%.

Figure 1. T-cell receptor knockout using TrueCut Cas9 Protein v2 and TrueGuide synthetic gRNAs. Human primary T cells were transfected with TrueCut Cas9 Protein v2 and TrueGuide Synthetic gRNA for T cell receptor alpha (TRAC) or beta (TRBC) regions using the Neon Transfection System. Editing efficiency was measured by (A) the GeneArt Cleavage Detection Assay, and (B) measuring % T cell receptor-negative (TCR^–) cells using the Attune NxT Flow Cytometer. By both measures, knockout efficiency exceeded 90% for TRAC and 80% for TRBC.

  Download the Human Primary T cell knockout application note

TrueCut Cas9 Protein v2 consistently performs across genes and cell lines.

Figure 2. Robust editing efficiencies in cancer cells. TrueGuide Synthetic gRNAs for three genes plus negative control were complexed with TrueCut Cas9 Protein v2; the RNP complexes were delivered into A549, U2OS, or MDA-MB231 cells using Lipofectamine CRISPRMAX Cas9 Transfection Reagent. At 72 hours post-transfection, cells were harvested and tested for cleavage efficiency (indel %) using either the GeneArt Genomic Cleavage Detection Assay (GCD) or Ion Torrent Next-Generation Sequencing (NGS). Cleavage efficiency was high across all genes and cell lines tested.

Figure 3. Consistent CRISPR editing efficiency across cell types and delivery methods. Four representative cell types including adherent cell lines (A549, HepG2), suspension cells (THP1), and primary immune cells (primary human T cells) were transfected via CRISPRMAX reagent or Neon electroporation system. RNP complexes comprised TrueCut Cas9 Protein v2 and TrueGuide positive or negative control sgRNA. Editing efficiency was high for TrueCut Cas9 Protein v2 across all cell types and methods tested.

TrueCut Cas9 Protein v2 consistently delivered up to two-fold higher gene editing efficiency compared to competitor Cas9 enzymes.

Figure 4. Invitrogen Cas9 compared to other Cas9 enzymes. Genome editing was performed with TrueCut Cas9 Protein v2 and corresponding TrueGuide Synthetic gRNAs. Complexes were delivered using optimized transfection protocols and Lipofectamine CRISPRMAX Transfection Reagent in (A) A549 and (B) MDA-MB231 cell lines. Cleavage efficiency was consistently greater with the Invitrogen system compared to products and protocols from other suppliers, even for challenging loci like PRKCG-T1 and CMPK1-T1.
 

The fidelity (specificity, or minimization of off-target effects) and editing efficiency (percentage of on-target edits) of TrueCut HiFi Cas9 Protein was assessed against TrueCut Cas9 Protein v2 and a competitor's high fidelity Cas9.  TrueCut HiFi Cas9 demonstrated superior ability to reduce measurable off-target edits.

For more detailed data including examples of both problematic and non-problematic loci, see our application note.
 

Figure 5. TrueCut HiFi Cas9 Protein demonstrated superior off-target profiles in human primary T cells. 21 gRNAs were co-transfected with wild-type TrueCut Cas9 Protein v2, TrueCut HiFi Cas9 Protein, and another supplier's 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. (A) The red dots are on-target events normalized to 100%. Gray 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. (B) Bars represent the number and severity of off-target edits according to off-target/on-target ratio level. Compared to wild-type TrueCut Cas9 Protein v2 and the competitor’s HiFi Cas9, TrueCut HiFi Cas9 Protein produced far fewer off-target effects at all severity levels.

In the same experiment, the on-target editing efficiency of TrueCut HiFi Cas9 equaled or exceeded that observed with the competitor’s high-fidelity Cas9, while also producing far fewer off-target effects.

Figure 6. TrueCut HiFi Cas9 Protein maintained >80% on-target editing efficiency in human primary T cells. On-target activity in T cells, including indels and homology-directed repairs (HDRs), was evaluated using a subset of 11 gRNAs that had the fewest off-target edits. Ion Torrent NGS-based Targeted Amplicon-seq was used for screening. Normalized to 100% for TrueCut Cas9 Protein v2, TrueCut HiFi Cas9 Protein achieved 84% and 88% editing efficiency for indels and HDRs, respectively.

In induced pluripotent stem cells (iPSCs), TrueCut HiFi Cas9 generated fewer off-target edits and had lower off/on ratios for individual off-targets than wt-Cas9 and the competitor’s high-fidelity Cas9. At the same time, TrueCut HiFi Cas9 Protein maintained high on-target editing activity with all gRNAs tested.

Figure 7. TrueCut HiFi Cas9 Protein demonstrated superior off-target profiles while maintaining high on-target editing efficiency in iPSCs.(A) The bar graphs show RPMs for on- and off-targets for the three genes in which off-targets were detected. TrueCut HiFi Cas9 achieved equivalent on-target efficiency and higher fidelity than that of wt-Cas9 and the competitor’s high-fidelity Cas9. (B) For both BCL11A and HBB1, the percentage of successful indels and HDRs achieved by TrueCut HiFi Cas9 Protein was comparable to both wild-type TrueCut Cas9 Protein v2 and the competitor’s HiFi Cas9.