Fig. 4: enGager mediated genome integration enhancement is ssDNA dependent. | Nature Communications

Fig. 4: enGager mediated genome integration enhancement is ssDNA dependent.

From: Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration

Fig. 4

A Schematic diagram of various Cas9-ssDNA binding protein and peptide fusion constructs (enGagers) in mRNA form. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein. GS is a shortened poly-GS peptide linker. B Schematic diagram of Knock in strategy of a 2Kb cssDNA donor template for RAB11A locus. C Dose titration of cssDNA from 0.3, 1, 1.5, 2 and 3ug for 2Kb GFP transgene knock-in at day 3 post electroporation. Cas9-RecA mRNA enhance around 25–30% knock in efficiency than Cas9 WT mRNA at all the cssDNA dose tested in k562 cells. D Representative FACS profiles with gating strategy showing % of 2Kb GFP transgene cassette Knock-in on RAB11 locus by cssDNA donor at day 5 post electroporation for various enGagers listed in (A) in K562 cells. E Quantification of 2Kb GFP transgene cassette Knock in fold change (left) and cell viability (right) of various enGagers as compared to Cas9 WT at day 5 post electroporation from (D). F Representative FACS profiles with gating strategy showing % of 2Kb GFP transgene cassette Knock-in on RAB11 locus by dsDNA donor at day 5 post electroporation for various enGagers listed in A in K562 cells. G Quantification of 2Kb GFP transgene cassette Knock in fold change (left) and cell viability (right) of various enGagers as compared to Cas9 WT at day 5 post electroporation from (F). H Quantification of 2Kb GFP transgene cassette Knock in fold change (left) and cell viability (right) of various enGagers mRNA as compared to Cas9 WT mRNA with cssDNA donor at 5 days post-delivery in HEK293 cells by lipofectamine 3000 transfection. I Quantification of 2Kb GFP transgene cassette Knock in fold change (left) and cell viability (right) of various enGagers mRNA as compared to Cas9 WT mRNA with dsDNA donor at 5 days post-delivery in HEK293 cells by lipofectamine 3000 transfection. Bars represents mean ± SD from 3 biological replicates. Illustrations for panels (E, G–I) are generated with BioRender.

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