Fig. 5: enGagers in mRNA form enhance genome integration on various locus and large payload.

A Schematic diagram of Knock in strategy of 2Kb (css76), 4Kb (css116) and 8Kb (css167) cssDNA donor templates for RAB11A locus. B Quantification of % of 2Kb (left), 4Kb (middle) and 8Kb (right) GFP transgene cassette Knock-in for various mRNA enGagers RAB11A day 13 post electroporation in K562 cells. C Quantification of cell viability for 2Kb (left), 4Kb (middle) and 8Kb (right) GFP transgene cassette Knock-in for various mRNA enGagers on RAB11A locus day 13 post electroporation in K562 cells. D Schematic diagram of Knock in strategy of 2Kb (css27), 4Kb (css88) cssDNA donor templates for B2M locus. E Quantification of % of 2Kb (left) and 4Kb (right) GFP transgene cassette Knock-in for various mRNA enGagers on B2M locus day 13 post electroporation in K562 cells. F Quantification of cell viability for 2Kb (left) and 4Kb (right) GFP transgene cassette Knock-in for various mRNA enGagers on B2M locus day 13 post electroporation in K562 cells. Bars represents mean ± SD from 3 biological replicates for (B, C, E, F). ***p < 0.001; ns non-significant, paired t-test compared to mRNA-Cas9 groups for C&E. G Schematic diagram showing enGager mRNA/sgRNA/cssDNA delivery into cells using LNP formulation. Once the editor components were delivered into the cytoplasm, the enGager mRNA is translated into endonuclease protein which forms a complex with sgRNA and cssDNA donor template. The assembled tripartite editing machinery complex then can be effectively shuttled into the nucleoplasm and tether onto the target genomic locus for transgene integration. Quantification of GFP transgene knock-in on RAB11A locus in HEK293 cells (H) and HepG2 cells (I) by LNP delivery at day 2, day 6 and day 9 post-delivery. Data were compared to mRNA-WT Cas9. Bars represents mean ± SD from 2 biological replicates. Illustrations for panel (G) is generated with BioRender.