Experiment: Imaging quantification of transfection efficiency with varying dosages of nanoparticles encapsulated with Cas9/sgRNA RNP on the liver-on-chip model system
PI: Ross C. Wilson, PhD Samira Kiani, PhD Shaoqin (Sarah) Gong, PhD
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Results |
Delivery Efficiency | |||||||
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Condition | Editor | Model | Delivery | Guide | Dosage | Corrected total fluorescence (mean gray value) | Image |
2.4µg RNP-NC-CPP TAT | sNLS-SpCas9-sNLS | Liver On Chip (LoC) | RNP-NC-CPP | Negative control gRNA | 2.4µg RNP | ||
2.4µg RNP-NC-tri(GalNAc) | sNLS-SpCas9-sNLS | Liver On Chip (LoC) | RNP-NC-tri(GalNAc) | Negative control gRNA | 2.4µg RNP | ||
24µg RNP-NC Lipo 1:1 | sNLS-SpCas9-sNLS | Liver On Chip (LoC) | RNP-NC-no ligand with Lipofectamine 2000 | Negative control gRNA | 24µg RNP | ||
24µg RNP-NC-CPP TAT | sNLS-SpCas9-sNLS | Liver On Chip (LoC) | RNP-NC-CPP | Negative control gRNA | 24µg RNP | ||
24µg RNP-NC-tri(GalNAc) | sNLS-SpCas9-sNLS | Liver On Chip (LoC) | RNP-NC-tri(GalNAc) | Negative control gRNA | 24µg RNP |
Transfection of higher dose (24ug) of RNP NCs in Primary Human Hepatocytes (PHH) shows higher uptake when compared to lower dose (2.4ug) |
A) Shows transfection of 24ug RNP-NC tagged with no ligand but labeled with Atto 550 in PHH when used in a 1:1 ratio with lipofectamine. B) Shows transfection of 24ug RNP-NC tagged with TAT (Cell Penetrating Peptide) labeled with Atto 550 in PHH. C) Shows transfection of 24ug RNP-NC tagged with TriGalNac ligand but labeled with Atto 550 in PHH D) Shows transfection of 2.4ug RNP-NC tagged with TAT ligand and labeled with Atto 550 in PHH. E) Shows transfection of 2.4ug RNP-NC tagged with TrigalNac ligand and labeled with Atto 550 in PHH |