Fig. 14. Exosome-based CRISPR transport. (a) A schematic illustration of exosome for in vivo delivery of Cas9 RNP for the treatment of liver disorders, (b) and exosomeRNP complexes (c), (d) biomarkers of exosome by western blotting. (e and f) DLS and TEM images of purified exosome. The arrows show the typical exosome nanoparticles. (f and G) DLS and TEM images of exosomeRNP complexes. The arrows show the typical exosomeRNP nanoparticles. (h and i) Cytosolic delivery of Cas9-FITC into LX-2 (h) and Huh-7 (i) cells by exosomes for 4 hours. The red arrows point at the efficient translocation of RNP into the nuclei. Scale bars, 25 μm. DAPI, 4′,6-diamidino-2-phenylindole. (j) Exosome-mediated Cas9 RNP delivery for genome editing. (k) Frequency of PUMA indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (l) Frequency of CcnE1 indel mutation detected by T7E1 assay from AML-12 cells after the specified treatments. (m) Frequency of KAT5 indel mutation detected by T7E1 assay from LX-2 cells after the specified treatments. (n) In vivo distribution of DiR-labeled exosomes in whole mice (top) or in the organs of the mice (bottom). H, heart; Lu, lung; Li, liver; K, kidney; and S, spleen. (o) A schematic illustrating the procedure to isolate different hepatic cell types and determine exosomeRNP biodistribution. (p) Percentage of each hepatic cell type that is DiI-labeled exosomeRNP-positive. (q) Relative MFI of each hepatic cell type. (r and s) Mechanism of cellular uptake of exosomeRNP nanocomplexes in LX-2 (r) and Huh-7 (s) cells by the addition of different inhibitors (reproduced with permission under Creative Commons CC BY 4.0 license from ref. 151 Copyright @ 2022 The Authors).