OptiLNP CRISPR Gene Editing Kit (Immune Cells) 

Lipid nanoparticles (LNP) are a lipid-based nanoscale drug delivery system widely used for the delivery of nucleic acids (such as mRNA, siRNA, DNA, etc.) and small molecule drugs.

Naked RNA is a negatively charged hydrophilic macromolecule that faces challenges in entering cells due to electrostatic repulsion from the cell membrane and is easily degraded by RNases. By encapsulating RNA within LNP, RNA can effectively traverse the cell membrane and be released into the cytoplasm, enabling efficient delivery.

MCE OptiLNP CRISPR Gene Editing Kit (Immune Cells) is a ready-to-use transfection reagent based on LNP technology. It utilizes the unique transmembrane transport and escape mechanism of LNP to achieve efficient delivery of RNA, making it suitable for co-transfection of Cas9 mRNA and sgRNA during CRISPR gene editing in immune cells.

Features of MCE OptiLNP CRISPR Gene Editing Kit (Immune Cells)

1. Lipid Nanoparticle (LNP) Technology: Utilizes LNP carriers to effectively protect RNA from degradation and immune system clearance during the delivery process.

2. Efficient Intracellular Delivery: The unique transmembrane transport and escape mechanisms of LNPs achieve an encapsulation rate of over 80% for small sample amount.

3. User-Friendly Operation: No encapsulation equipment is required; RNA-LNP encapsulation and transfection can be completed in a single mixing step.

4. Biocompatibility: Low cytotoxicity, effectively balancing high transfection efficiency with cell viability, and providing gentle action

5. Safety: The formulation is ethanol-free and uses biodegradable lipid materials to ensure safety.

Transfection Reagent, Dilution Buffer: 4℃, 1 year. Do not freeze.

Cas9 mRNA (Lyophilized), sgRNA Positive Control (Lyophilized), and sgRNA Positive Control Primers: -20℃, 1 year

1. This product has been filtered using a 0.22 μm membrane.

2. The Cas9 mRNA and sgRNA Positive Control is provided as a lyophilized powder. Please centrifuge before opening the cap. Use nuclease-free water to dissolve and prepare a positive control stock solution at 1 μg/μL. The stock solution can be stored at -80°C for 6 months, avoiding repeated freeze-thaw cycles.

3. For optimal transfection efficiency, it is recommended to dissolve the RNA stock in nuclease-free sterile water. Other solvents may affect transfection efficiency. It is recommended to use commercially available RNA; for self-prepared RNA, purification and desalting are necessary to prevent RNA precipitation.

4. It is recommended to prepare RNA pre-mixes using the dilution buffer provided with the kit to dilute the RNA stock solution.

5. RNA-LNP complexes can be stably stored at room temperature for up to 8 h. Do not store RNA-LNP complexes under freezing conditions.

6. If dilution of the RNA-LNP complex is required, it is recommended to use the dilution buffer provided with the kit for dilution.

7. The RNA/LNP ratio is crucial for LNP encapsulation. If the per-well RNA amount needs adjustment, the volume of RNA-LNP complex added to each well should be proportionally adjusted.

8. When co-transfecting Cas9 mRNA and sgRNA, the RNA pre-mix concentration refers to the total concentration of Cas9 mRNA and sgRNA. The recommended ratio is: μgmRNA : μg sgRNA = 1:1 - 1:2.

9. There is no need to replace the culture medium before or after transfection. Complete medium containing serum and antibiotics does not significantly affect transfection efficiency.

10. 6 h after transfection, standard cell culture processes, such as medium replacement, can be performed.

11. Cell conditions greatly influence transfection efficiency. It is recommended to use cells in good growth condition for transfection.

12. There are many factors

Take the 24-well plate as an example; the cell volume of other culture well plates can be designed according to the conventional experiments.

For suspension cells: Plate the cells before transfection and suspend in fresh medium (0.5 - 2.5 × 10⁵ cells/500 μL medium).

Note: The viability and general health of cells prior to transfection significantly affect the transfection result. Cells should be at least 90% viable prior to transfection and have had sufficient time to recover from passaging.

1) Prepare a 1 μg/μL stock solution of Cas9 mRNA (lyophilized) using nuclease-free water.

2) Prepare a 1 μg/μL stock solution of sgRNA positive control (lyophilized) using nuclease-free water.

Refer to the table below, take the appropriate amount of Cas9 mRNA stock solution and sgRNA positive control stock solution, and dilute the stock solutions with dilution buffer to achieve a total RNA concentration of 40 ng/μL.

Refer to the table below. Mix equal volumes of transfection reagent and RNA pre-mix directly, and pipette up and down at least 20 times to ensure thorough mixing.

Add RNA-LNP Complex to each well, mix gently and incubator for further culture.

After incubation of 24 - 48 h, the transfection effect can be analyzed by fluorescence detection, Western Blot, ELISA, flow cytometry, reporter gene, immunofluorescence staining and other methods according to the experimental needs.

1. This product has been filtered using a 0.22 μm membrane.

2. The Cas9 mRNA and sgRNA Positive Control is provided as a lyophilized powder. Please centrifuge before opening the cap. Use nuclease-free water to dissolve and prepare a positive control stock solution at 1 μg/μL. The stock solution can be stored at -80°C for 6 months, avoiding repeated freeze-thaw cycles.

3. For optimal transfection efficiency, it is recommended to dissolve the RNA stock in nuclease-free sterile water. Other solvents may affect transfection efficiency. It is recommended to use commercially available RNA; for self-prepared RNA, purification and desalting are necessary to prevent RNA precipitation.

4. It is recommended to prepare RNA pre-mixes using the dilution buffer provided with the kit to dilute the RNA stock solution.

5. RNA-LNP complexes can be stably stored at room temperature for up to 8 h. Do not store RNA-LNP complexes under freezing conditions.

6. If dilution of the RNA-LNP complex is required, it is recommended to use the dilution buffer provided with the kit for dilution.

7. The RNA/LNP ratio is crucial for LNP encapsulation. If the per-well RNA amount needs adjustment, the volume of RNA-LNP complex added to each well should be proportionally adjusted.

8. When co-transfecting Cas9 mRNA and sgRNA, the RNA pre-mix concentration refers to the total concentration of Cas9 mRNA and sgRNA. The recommended ratio is: μgmRNA : μg sgRNA = 1:1 - 1:2.

9. There is no need to replace the culture medium before or after transfection. Complete medium containing serum and antibiotics does not significantly affect transfection efficiency.

10. 6 h after transfection, standard cell culture processes, such as medium replacement, can be performed.

11. Cell conditions greatly influence transfection efficiency. It is recommended to use cells in good growth condition for transfection.

12. There are many factors