Figure 3.

RNP accounted for the gene editing activity. (A) HBB sgRNA1 expressed from transfected plasmid DNA was functional. In co-transfection experiments, the DNA amount indicated each of the Cas9 expressing- and the sgRNA expressing-plasmid DNA. Each point was the average of three replicates. (B) Importance of Cas9 co-packaging and com-aptamer modification of sgRNA on gene editing activity of the LVLPs. LVLPs packaged in the absence of Cas9 expression were inactive when co-transduced into GFP-reporter cells with functional Cas9-HBB-3′UTR^MS2 mRNA LVLPs. About 2.5 × 10⁴ GFP-reporter cells were transduced with indicated particles. GFP-positive cells were determined by flow cytometry 48 h after transduction. Each point is the average of three replicates. *** indicates P < 0.001 when GFP-positive rates of cells treated with com⁺ RNP LVLPs were compared with cells treated with similar amounts of other particles (Bonferroni posttests following ANOVA). (C) Co-expressing Cas9 increased HBB sgRNA1 level. Plasmids expressing only HBB sgRNA1^Tetra-com (200 ng) and only SaCas9 (200 ng) were transfected into HEK293T cells alone or together, and the sgRNA level was compared by RT-qPCR. A GFP-expressing plasmid (50 ng) was co-transfected so that GFP expression could be used to normalize transfection efficiency. Total plasmid DNA was brought to 450 ng by pCDNA3 plasmid DNA. * indicates P < 0.05 when sgRNA level without Cas9 co-expression was compared with that of with Cas9 co-expression. (D) Western blotting analysis of Cas9 protein in isolated lentiviral vectors and LVLPs. About 200 ng p24 of GFP lentivirus (lane 1), NC-MCP modified Cas9-HBB-3′ UTR^MS2 LVLPs (without sgRNA, lane 2), NC-unmodified Cas9^MS2 LVLPs (without sgRNA, lane 3), NC-COM modified Cas9/HBB sgRNA1^tetra-com LVLPs (lane 4), NC-COM modified Cas9/IL2RG sgRNA1^tetra-com LVLPs (lane 5) and NC-COM modified Cas9/HBB sgRNA1 LVLPs (sgRNA without Tetra-com aptamer, lane 6) were loaded. (E) Tetra-com modification of sgRNA increased the Cas9 protein content in LVLPs. (F) Cas9 proteins in LVLPs with com-modified sgRNA are more detergent-resistant than Cas9 proteins in LVLPs with unmodified sgRNA. The same amount of starting LVLPs (200 ng of p24) was centrifuged through 1 ml of 10% sucrose with or without 0.5% Triton X-100. For panels (E) and (F), Cas9 level was normalized by CA protein, based on dosimetry analysis (IMAGE J). (G) Packaging of sgRNA in LVLPs is com-aptamer but not Cas9 protein dependent. See panel (E) for evidence of similar particle input (CA) for RNA isolation. Each point indicates one repeat. *** indicates P < 0.001 between the indicated pairs in Tukey’s Multiple Comparison Test following ANOVA analysis.