Description
The LNP formulation loaded with eSpCas9 mRNA (m1Ψ) and TRAC sgRNA is prepared using generic LNP formulation with main ionizable lipid of SM102.
The payload eSpCas9 mRNA (m1Ψ) has all the U bases 100% modified with N1-methyl-pseudouridine (m1Ψ). HPLC purified SafeEdit sgRNA with modifications targeting the first exon of the constant chain of the TCRα gene (TRAC), which enhances CAR-T cell potency and persistence by utilizing the endogenous transcriptional control of TCR gene.
The eSpCas9 mRNA and TRAC sgRNA are loaded at a molar ratio of 1:10 in this LNP formulation.
This formulation is a good control for testing if SM102 LNP formulation is a good potential formulation for your CRISPR gene know out product under research in your in vitro and in vivo experiment model. |
| Form |
Liquid |
| Concentration |
0.15mg/mL |
| Full mRNA length |
4471nt |
| Full mRNA Molecular Weight |
1457890 |
| Storage buffer |
Tris-HCl/Sucrose |
| Storage condition |
Store at -80°C for long term. Avoid freeze thaw, only thaw once before use. |
| Appearance |
Clear and free of foreign particles |
| Encapsulation Efficiency |
> 85% |
| Endotoxin |
< 4 EU/ml |
| Particle Size |
65-125 nm |
| pH |
7.4 ± 0.5 |
| Polydispersity Index |
< 0.2 |
| Zeta Potential |
± 15.0 mV |
Guide for LNP-CRISPR gene knockout (KO) operation of Jurkat cells:
1. Cell culture
- Change the culture medium every 2-3 days.
- Passage the cells 2-3 times per week. The recommended cell passage ratio is 1:5 - 1:6.
- Maintain the cell density during culture at 2×105 - 1.5×106 cells/ml.
2. LNP transfection (24-well plate)
- On the day of transfection, perform cell counting to determine the culture density. Add 0.5 ml of complete growth medium to each well, and plate with 1 x 105 cells. On the day of transfection, the cell density should reach ~80% confluence.
- Directly add LNP containing 0.5μg Cas9 mRNA & sgRNA to each cell well, and gently shake the culture plate back and forth to mix.
- After transfection, incubate the cells in a 37°C CO2 cell culture incubator for 2-3 days for gene knockout analysis. |
For laboratory research use only. Direct human use, including taking orally and injection and clinical use are forbidden.
