Recombinant stable HEK293 cell line constitutively expressing a FLAG-tagged Cas9 nuclease and GFP (Green Fluorescent Protein), which have been stably integrated into the AAVS1 safe harbor locus on chromosome 19. When transfected or transduced with single guide RNAs (sgRNAs), this cell line will sustain double-strａnd DNA breaks (DSBs) at targeted genome sites.

Expression of Cas9 is driven by a CBh promoter, whereas GFP is driven by an EF1A promoter. The combined DNA fragment (CBh-Cas9-bGH Poly A-EF1A-GFP-T2A-Puro-SV40 Poly A), shown in Figure 1, was integrated at the AAVS1 safe harbor locus using CRISPR/Cas9 technology. The construct also contains a puromycin resistance gene.

Cells were cloned by limiting dilution to obtain a monoclonal population. Without the adverse effects resulting from random integrations of Cas9/GFP into the HEK293 genome, this cell line behaves like parental HEK293 cells.

Figure 1: Transgene integration at the AAVS1 locus.

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