Fig 1.
Features of CRISPR/Cas RNP delivery methods suitable for development of cell-based therapies and those considered ‘research-use only’.
Fig 2.
Example of transfection plate layout.
Experiments where (A) optimal ratio of gRNA to CRISPR/Cas protein required for precomplexing will be determined, (B) the optimal amount of recombinant CRISPR/Cas will be determined (if we assume the optimal ratio will be 1:1) and (C) optimization of transfection reagent amount (1×, 2× and 3×) used for complex formation (if we assume the optimal CRISPR/Cas amount is 0.6 pmol per well). Wells named “T.R.” will contain transfection reagent only. White wells will hold non-transfected cells used as non-treated controls for the cell viability assay. The crossed-out wells represent empty wells used for holding the PrestoBlue™ Cell Viability Reagent staining blank. Grey wells will be filled with PBS to avoid edge effect. All experiments will be performed in triplicate, 4 gRNAs will be used for RNP formation (AAVS1, CDK4 and HPRT1 and non-targeting control gRNA).
Fig 3.
Example of 20 μl Nucleocuvette™ Strip layout.
Experiments where (A) optimal ratio of gRNA to CRISPR/Cas protein required for precomplexing will be determined, (B) the optimal amount of recombinant CRISPR/Cas will be determined (if we assume the optimal ratio will be 1:1). All experiments will be performed in duplicate, 4 gRNAs will be used for RNP formation (AAVS1, CDK4 and HPRT1 and non-targeting control gRNA).
Table 1.
CRISPR/Cas RNP delivery to immortalized cell lines and primary human cells using 4D-NucleofectorTM System.
Table 2.
CRISPR/Cas RNP delivery to target cells using Neon™ Transfection System.
Table 3.
NaCl and GABA concentrations optimization during iTOP protein transduction (48-well transduction plate layout).
Table 4.
Osmoprotectants concentrations optimization during iTOP protein transduction (48-well transduction plate layout).
Table 5.
Betaine and glycerol concentrations optimization during iTOP protein transduction (48-well transduction plate layout).
Table 6.
Proline and glycerol concentrations optimization during iTOP protein transduction (48-well transduction plate layout).
Table 7.
Glucose, sucrose and mannitol concentrations optimization for GSM protein transduction.
Table 8.
Sucrose and PEG1000 concentrations optimization during standard osmotic lysis of pinocytic vesicles technique (48-well transduction plate layout).