Fig. 2: Identification of mini enGagers with Cas9 fused ssDNA binding motifs in K562 cells. | Nature Communications

Fig. 2: Identification of mini enGagers with Cas9 fused ssDNA binding motifs in K562 cells.

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

Fig. 2: Identification of mini enGagers with Cas9 fused ssDNA binding motifs in K562 cells.

A Schematic diagram of various Cas9-ssDNA binding motifs fusion constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein. Cas9-RecA fusion construct was used as a positive control. FECO, WECO and YECO are 20 amino acid sequences previously identified as ssDNA binding motifs in various bacteria species of RecA(Voloshin et al., 1996), FECO3X, WECO3X and YECO3X are 3 tandem copies of the 20 aa peptides separated by multi-GS peptide linkers. B Schematic diagram of Knock in strategy of a 2Kb cssDNA donor template for RAB11A locus. C Representative FACS profiles with gating strategy showing % of GFP transgene cassette Knock in on RAB11 locus at day 7 post electroporation for various small enGagers listed in (A). D Quantification of 2Kb GFP transgene cassette Knock in fold change of various enGagers as compared to Cas9 WT at day 7 post electroporation. Note that Cas9-FECO fusion performs similarly with Cas9-RecA fusion in cssDNA mediated transgene integration (1.59- vs 1.58-fold). EnGagers with 3X tandem ssDNA binding peptides do not further enhance knock in efficiency does not enhance knock in efficiency. Bars represent mean ± SD from 3 biological replicates. Illustrations for panel C is generated with BioRender.

Back to article page