Fig. 3: Identification of additional enGagers from Cas9-ssDNA binding module chimeras in K562 cells. | Nature Communications

Fig. 3: Identification of additional enGagers from Cas9-ssDNA binding module chimeras in K562 cells.

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

Fig. 3

A Schematic diagram of various Cas9-ssDNA binding protein and peptide 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 and Cas9-FECO fusion constructs were used as positive controls. The fusion protein or peptides include DrRecA, 20 aa motif identified from DrRecA, SSAP, Lambda Red, RecT, RadA and RadB from Archaea. B Schematic diagram of Knock in strategy of a 2Kb cssDNA donor template for RAB11A locus. C 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-DrRecA and Cas9-DrRecA20AA fusion has the highest performance in knock in with 2.17- and 2.43-fold as compared to Cas9 WT, respectively. **p < 0.01, paired t-test compared to AIO Cas9 group. D Quantification of cell viability day 7 post electroporation. Bars represents mean ± SD from 3 biological replicates for (C, D). E Amino acid sequence alignment of 20AA of multiple E.coli RecA mutant variants and RecA from archaea and mammalian organism. Dr Deinococcus radiodurans, Ec Escherichia coli, Sc Saccharomyces cerevisiae, Hs Homo sapiens, Pf P. furiosus, Sso S. solfatarcus.

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