Figure 3.

4A3-SC8 dLNPs successfully induced HDR in HEK293 cells containing a GFP sequencing with a Y66H mutation via one-pot delivery of Cas9 mRNA, sgRNA, and a corrected ssDNA template. A) HEK293 cells contain a single amino acid mutation in their GFP sequence (Y66H) that alters their fluorescence. Depending on the gene editing technique employed, the fluorescence can be eliminated (NHEJ) or restored to native GFP (HDR). B) HEK293 (Y66H) cells were transfected with dLNPs containing 1000 ng of only Cas9 mRNA and sgRNA at a 2:1 ratio or dLNPs containing Cas9 mRNA, sgRNA, and ssDNA template at a 2:1:1 ratio and imaged using confocal microscopy for mutated GFP (blue) and GFP (green) signal. CellMask Orange was used to stain plasma membranes in merged images. C–E) The amount and type of gene editing induced were assessed using TIDER to analyze DNA sequencing following transfection with HDR dLNPs containing Cas9 mRNA:sgRNA ratios fixed at 1:1 (C), 1:2 (D), and 2:1 (E) (N = 3). F) Across all three fixed ratios of Cas9 mRNA:sgRNA similar trends in the amount of HDR, NHEJ, total efficacy, and unedited cells were observed, with the 2:1 Cas9 mRNA:sgRNA exhibiting the highest degree of all forms of editing (N = 3).