Gene Editing Enzymes & Reagents

Explore our GMP-Grade CRISPR Cas9, Cas12a, MaxNuclease, AAV ELISA Kits, and DNA Amplification Enzymes

Overview

Gene therapy generally functions by introducing exogenous genes (or gene-editing tools) into target cells or tissues, serving to replace, compensate, block, correct, augment, or knockout specific genes to exert therapeutic effects [1]. Based on the different methods of introducing genes into the human body, gene therapy can be divided into ex vivo therapies (extracorporeal therapies) and in vivo therapies (intracorporeal therapies). Ex vivo therapy generally involves introducing exogenous genes (or gene-editing tools) into cells outside the body, preparing them to become genetically modified cells or cell-derived products, and ultimately reintroducing them to the body to exert therapeutic effects. In vivo therapy introduces exogenous genes (or gene-editing tools) into the human body via non-viral vectors (such as LNP) or viral vectors (such as Adeno-Associated Virus AAV, Lentivirus LV, etc.) to exert therapeutic effects.

Figure 1. Types of gene editing [1][2].

GMP Cas9 & Cas12a Nucleases

Cas9 and Cas12a are primarily used for the genetic modification of cells and gene therapy drugs, including hematopoietic stem cells and T cells. Our GMP-Grade Cas9 nuclease is manufactured in a high-standard GMP-Grade enzyme production facility and has undergone rigorous in vitro and intracellular quality validation. KACTUS has filed our GMP Cas9 with the FDA Drug Master Files (DMF# 036578). KACTUS has provided regulatory support to customers completing Investigational New Drug (IND) applications with our Cas9.

Figure 2. (Left) Cas9 Nuclease is used for gene knockout in 293T cells, Jurkat cells, and T cells, and its gene knockout efficiency in various cell types is comparable to that of leading suppliers. (Right) Cas9 Nuclease In Vitro DNA Cleavage Experiment. The in vitro cleavage efficiency of Cas9 nuclease is equivalent across all three batches.

Cas9 ELISA Kit

KACTUS Cas9 ELISA kit is for quantitative detection of residual Cas9 nuclease in gene-edited cell drugs before reinfusion into the human body, whether extracellular or intracellular. Our test kit has a detection range of 0.25 ng/mL to 16 ng/mL, with sensitivity reaching up to 0.125 ng/mL.

High Accuracy and Standard Curve R² > 0.99

Figure 3. (Left) Cas9 ELISA Kit example standard curve with an R² of 0.9997. (Right) The same test kit was used to assess the spike-recovery rate of Cas9 nuclease at three different concentrations (high, medium, low) in dilution buffer. The spike-recovery rate for different concentrations of Cas9 samples in dilution buffer all fell between 80%-120%, indicating that this ELISA kit has high accuracy in detecting residual Cas9 nuclease.

GMP-Grade DNA/RNA Degradation with MaxNuclease

In gene therapy, MaxNuclease principally serves to eliminate contaminants like host DNA and plasmid DNA present during the pharmaceutical production process. This GMP-grade nuclease is produced within a facility maintaining stringent GMP-Grade production standards, avoiding the use of materials derived from animals. The analytical methods applied have undergone systematic validation, and the final enzyme is subject to extensive quality control release testing to ensure it meets the requirements for use from research and development through to large-scale commercial production. Additionally, KACTUS has submitted MaxNuclease to the FDA Drug Master Files (DMF #036799).

Figure 4. Virus harvest solution was treated with 25U/mL and 50U/mL endonuclease at 37°C for 2 hours, respectively. Detection of plasmid DNA (pDNA) residue (left) and host cell DNA (HCD) (right) residue was analyzed. KACTUS MaxNuclease has higher degradation activity versus Competitor B demonstrated by lower pDNA and HCD residue for both 25U/mL and 50U/mL working concentrations.

MaxNuclease ELISA Kit

The quantification of residual MaxNuclease in gene therapy drugs is achievable with our associated ELISA kit, which provides a wide detection range of 46.88pg/mL to 3000pg/mL and sensitivity reaching up to 23.44pg/mL.

Figure 5. Example standard curve for MaxNuclease ELISA kit.

ELISA Kits for Residual AAV Quantification

AAV is a commonly used viral vector in the field of gene therapy. Methods for detecting the titer of AAV viral particles include Dot blot, TEM, AUC, ELISA, SEC-MALS, etc. KACTUS has developed an AAV5 and AAV9 Titration ELISA Kit, aiming to provide a more convenient and reliable solution for the quantification of residual AAV viral particle titer.

Figure 6. Example standard curve for the AAV9 Titration ELISA Kit, with a quantification range of 2.03E+07-1.30E+09 capsids/mL.

Specificity

Figure 7. (Left) An indirect ELISA was used to detect the cross-binding of different concentrations of AAV9 antibodies with AAV2/5/8/9. A curve with antibody concentration as the x-axis and OD450 value as the y-axis is given. Results show that AAV9 detection antibodies only specifically bind to AAV9 and do not cross-react with the capsid proteins of AAV2/5/8.

(Right) A sandwich ELISA was used to detect the binding of AAV9 antibodies with both intact and denatured AAV9. A curve with AAV9 capsid titers as the x-axis and OD450 value as the y-axis is given. Results show that AAV9 detection antibodies do not bind to the denatured AAV9 capsid.

Accuracy

Figure 8. The same AAV9 kit was used to test the spiked recovery rate of high, medium, and low titer AAV9 samples in dilution buffer. The spike recovery rates of different titer AAV9 samples tested in dilution buffer are all between 80% and 120%, indicating that the kit has high accuracy in detecting AAV9 titers.

In Vitro Enzymatic Synthesis of DNA Vectors

The enzymatic synthesis of DNA vectors in vitro can effectively replace traditional plasmid DNA production, perfectly avoiding the many uncontrollable risks of the biological fermentation process, and can complete industrial production in a shorter time, achieving the goal of accelerating production and reducing costs. In the field of gene therapy, it is mainly used to prepare viral vectors such as AAV (Adeno-Associated Virus), LV (Lentivirus), etc.

TelN Protelomerase

TelN Protelomerase can be used to synthesize linear, closed mini DNA vectors. It cuts double-stranded DNA at the recognition site TelRL (56bp) and leaves a covalently closed hairpin structure at the enzyme cutting site, effectively converting circular DNA into linear DNA with hairpin ends. In practical applications, the target fragment and the backbone sequence on the plasmid are separated by TelRL sites.

Note: In some application scenarios, the use of this enzyme may be subject to patent restrictions.

Figure 9. TelN Protelomerase can effectively convert circular DNA into linear DNA with hairpin ends. The activity of KACTUS TelN Protelomerase is comparable leading suppliers. In a 50 μL system, 323 fmol of dsDNA was added to 1 μL of TelN Protelomerase (wells 1-6 diluted in a 2-fold gradient respectively, with NC as the control group) in a 30 minute reaction at 37℃.

Phi29 DNA Polymerase

Phi29 DNA polymerase has strong strand displacement activity and characteristics of continuous synthesis, and can be used for synthesizing double-stranded DNA in vitro.

Note: The use of this enzyme may be subject to patent restrictions in some application scenarios.

ExoNuclease III & T5 Exonuclease

The DNA vectors formed by the cutting and linking of TelN Protelomerase are of two types: one contains the backbone sequence, and the other contains the target fragment. The backbone sequence has corresponding restriction enzyme cutting sites, forming blunt or sticky ends after enzymatic cutting. Further action by Exonuclease III or T5 Exonuclease achieves the degradation of the backbone sequence, serving the purpose of enriching the target fragment.

Figure 10. Exonuclease III & T5 Exonuclease effectively degrade double-stranded DNA linearized by BsaI (forming 5’ overhang ends after BsaI digestion), but have no effect on double-stranded DNA linearized by TelN Protelomerase (TelN Protelomerase linearization forms linear DNA with hairpin ends). In a 20 μl reaction system, add 2 μg of double-stranded DNA treated with BsaI/TelN Protelomerase and 1 μl of Exonuclease III/2 μl of T5 Exonuclease, and react at 37℃ for 30 min.

Note: In the figure, Exo III represents Exonuclease III; T5 Exo represents T5 Exonuclease.

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References

[1] Technical Guidance Principles for the Pharmacological Research and Evaluation of In Vivo Gene Therapy Products (Trial)

[2] Savić N, Schwank G. Advances in therapeutic CRISPR/Cas9 genome editing. Transl Res.