Fig. 2

CRISPR-Cas9 and insertion of F9 cDNA into AAVS1 locus of HEK293T cells. a Schematic of CRISPR-Cas9. b About 80% of cells were GFP-positive 24 h after transfection in the GFP group. c Primers F1, R1 were used to detect the full fragment of insertion. In the 293 T-WT group, only a 468-bp fragment could be seen (yellow arrow); in the 293 T-insertion group, both 468-bp and 4.9-kb fragments could be seen (yellow arrow). d. Primers F2, R2 used to detect 5′ junction point of insertion. In the 293 T-WT group, nothing could be seen; in the 293 T-insertion group, a 1.3-kb fragment could be seen (yellow arrow). e qRT-PCR showed F9 expression of the 293 T-insertion group was extremely higher than that in the 293 T-WT group. AAVS1-EF1α-puromycin empty plasmid did not produce any hFIX expression (n = 3 independent experiments for each sample). f Immunofluorescence staining showed hFIX expression in the 293 T-insertion group. All scale bars represent 100 μm. CRISPR-Cas9 clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9, WT wildtype, GAPDH glyceraldehyde 3-phosphate dehydrogenase, DAPI 4′,6-diamidino-2-phenylindole, HFIX human factor IX