Fig. 1

Development of the CRISPR–Cas9 knock-in assay. A Donor plasmid design for unmatched end joining strategies. The AAVS1 locus is targeted by CRISPR–Cas9 using the AAVS1-A1 guide. Cas9-targeted cleavage will form DNA DSBs at which the transgene is to be inserted. The unmatched end donor plasmid contains the transgene, which is a splice acceptor—driven EGFP coupled with the pac puromycin resistance gene for further selection. On the backbone, BFP expression is driven by a CAG promoter so as to report backbone integration. The donor plasmid contains Tet2 guide cleavage sites on either side of the transgene but does not have any sequences that are homologous to the AAVS integration site. B Overview of the knock-in assay. Three plasmids are transfected into HEK293 cells (i) Cas9 (wt or mutant) and mAmetrine expression and (ii) expression of sgRNAs for AAVS1 and Tet2, (iii) the unmatched end donor plasmid. Initial assessment by flow cytometry quantifies mAmetrine and BFP expression for transfection efficiency, and GFP to evaluate knock-in efficiency. Puromycin selection is then applied to enrich for cells in which the transgene is integrated and expressed. Final flow cytometry assessment is performed to collect GFP and BFP expression data to evaluate knock-in fidelity. C, D Data from the knock-in assay using wtCas9 and the unmatched end donor plasmid. C Flow cytometry quantification of GFP expressing cells 2 days after transfection. The donor plasmid only control was not transfected with the Cas9 and sgRNA plasmids. D Fluorescence profile of puromycin resistant cells after transfection and selection according to the unmatched end joining knock-in strategy. Error bars in C and D represent the standard error mean from three independent replicates. Students’ T tests indicate no significant differences between the donor only control and the unmatched end joining experiment. p > 0.05