Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes

Figures

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  FIGURE 1. Schematic illustration of EV‐mediated CRISPR/Cas9 RNP delivery knocking out Pcsk9 leads to increased LDLR recycling and LDLR expression on the cell surface. (a) PCSK9 binds the LDLR/LDL‐C complex and marks it for degradation by the lysosome. In the absence of PCSK9, the LDLR is free to bind and process plasma LDL‐C, whereafter, it is recycled back to the membrane surface to clear more LDL‐C from circulation. (b) Upon extracellular vesicle‐mediated CRISPR/Cas9 delivery, knock‐out of the Pcsk9 gene would reduce the LDLR/LDL‐C complex's degradation and increase LDLR recycling.

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  FIGURE 2. EVs mediate CRISPR/Cas9 RNP delivery in vitro (a) Schematic illustration of N‐Myr‐2xFKBP12/Cas9‐FRB construct and (b) Cas9‐EV production by transiently transfecting HEK293FT cells with N‐Myr‐FKBP12/FRB‐Cas9, VSV‐G and gRNA plasmids followed by differential ultracentrifugation. (c) NTA showed Cas9 EVs had a size distribution between 50‐250 nm with a mean peak size of 113,2 ± 2,3 nm. (d) TEM images showed Cas9 EVs had an average size around 100 nm. Scale bar = 100 nm (E) Western blot analysis demonstrated Cas9 EVs are enriched with Cas9 protein, FKBP12, VSV‐G and are positive for EV marker TSG101, CD63, CD81, synthenin‐1 and β‐actin. (f) Schematic illustration of CRISPR/Cas9‐mediated NHEJ leading to eGFP+ expression in Cas9 stoplight reporter cells. Administration of Cas9 EVs demonstrated successful CRISPR/Cas9‐mediated genome editing in Cas9 stoplight reporter cells visualized with (g) fluorescence microscopy analysis and (H) quantified by flow cytometry analysis with an efficiency of ±28,1%. Representative images of four individual experiments. Data presented as Mean ± SEM, analyzed using t‐test with *p < 0,05 and **p < 0,01. Scale bar = 200 μm.

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  FIGURE 3. Cas9 EV mediated delivery of CRISPR/Cas9 successfully targets Pcsk9 in mouse hepatocytes leading to increased LDLR levels and LDL‐C uptake (a) Schematic illustration of MMgRNA targeting the Pcsk9 gene (b) Agarose gel of T7E1‐treated PCR products amplified from lentivirus treated NIH3T3 cells show NHEJ frequencies for the different gRNA's (c) Quantification of on‐target indel mutation frequency by respective MgRNA's from lentivirus transduced cells (d) Agarose gel of T7E1‐treated PCR products amplified from mouse hepatocytes treated with EVs carrying either NTgRNa, MMgRNA 5, 8 or 9 (D) Quantification of NHEJ activity of Cas9 EVMMgRNA treated mouse hepatocytes (e) RT‐qPCR of relative Pcsk9 mRNA levels showed MMgRNA 9 successfully targets the exon splicing donor slice thereby interfering with Pcsk9 pre‐mRNA processing. (f) Western blot analysis was conducted on Cas9 EV‐treated mouse hepatocytes using antibodies against LDLR, PCSK9, and β‐actin. (g) Quantification of LDLR levels, normalized to β‐actin, demonstrated that administration of Cas9 EVs loaded with gRNA promoted the expression of the mature form of LDLR. (h) Quantification of intracellular PCSK9 levels, also normalized to…