General CRISPR/gRNA transfection guidelines
- The efficiency with which mammalian cells are transfected with gRNA varies according to cell type and the transfection reagent used. See Table 1 for delivery reagent recommendations.
- For gene editing we find highest editing efficiency with 1:1 molar ratio of gRNA to TrueCut Cas9 Protein v2. In some cell types such as iPSC and THP1, we have used up to 2 μg TrueCut Cas9 Protein v2 and 400 ng gRNA per well in 24-well format.
- The optimal cell density for transfection varies depending on cell size and growth characteristics. In general, we recommend 30–70% confluence on the day of transfection when using lipid-mediated delivery or 70–90% confluence for electroporation using the Neon Transfection System.
- After you have determined the optimal cell number and dosage of TrueCut Cas9 Protein v2/gRNA that provides maximal gene editing efficiency, do not vary these conditions across experiments for a given cell type to ensure consistency.
For an overview of the factors that influence transfection efficiency, refer to the “Transfection Basics” chapter of the Gibco Cell Culture Basic Handbook, available at thermofisher.com/cellculturebasics.
- Use the TrueGuide Positive Controls (human AVVS1, CDK4, HPRT1, or mouse Rosa 26) and negative control gRNA (non-coding) to determine gRNA amount and transfection conditions that give the optimal gene editing efficiency with highest cell viability. The TrueGuide Positive and Negative sgRNA and crRNA Controls are available separately from Thermo Fisher Scientific. For more information, refer to thermofisher.com/trueguide.
- The cell number and other recommendations provided in the following procedures are starting point guidelines based on the cell types we have tested. For multiple wells, prepare a master mix of components to minimize pipetting error, then dispense the appropriate volumes into each reaction well. When making a master mix for replicate wells, we recommend preparing extra volume to account for any pipetting variations.
Recommended delivery options
- Choosing the right delivery reagent is critical for transfection and gene editing efficiency. See our recommendations in Table 1. For more information on transfection reagents, see thermofisher.com/transfection.
- Cell line–specific conditions are provided in the tables: Cell line–specific transfection conditions using the Lipofectamine CRISPRMAX Transfection Reagent and Cell line–specific CRISPR RNP electroporation conditions using Neon System.
Table 1. Recommended delivery options for TrueCut Cas9 Protein v2.
|Cas9 format||Transfection reagent*||Electroporation**|
|TrueCut Cas9 Protein v2 + gRNA||Lipofectamine CRISPRMAX Cas9 Transfection Reagent||For maximum efficiency in difficult-to-transfect cell types, use the Neon Transfection System|
|* For best results, we recommend that you transfect your cells with TrueCut Cas9 Protein v2 + TrueGuide Synthetic gRNA (crRNA:tracrRNA duplex or sgRNA) using the Lipofectamine CRISPRMAX Cas9 Transfection Reagent.|
** Use the Neon Transfection System 10 μL Kit (Cat. No. MPK1025).