Fig. 3
The design of single-stranded oligodeoxynucleotides (ssODNs) and plasmid donor, and the comparison of HDR efficiencies between TALEN and CRISPR/Cas9. a The schematic diagram shows two 90-mer ssODN and a plasmid donor DNA used to incorporate the two substitutions (199T > C) and (437A > T) into the EGFP locus. Predicted cleavage sites of CRISPR/Cas9 or TALEN are indicated by coloured arrow heads. b The set up of controls for FACS analysis. HEK293FT cell line was used as EGFP and EBFP negative control. HEK293FTEGFP cell line was used as EGFP positive and EBFP negative control. HEK293FT cells transiently transfected with a plasmid encoding EBFP were used as EBFP positive control. In each case, a total of 10,000 events were counted. c Representatives of the FACS analysis of EBFP positive cells generated via HDR by using TALEN or CRISPR/Cas9 with donor templates. NG: EGFP negative cells. d Detection of targeted genomic deletions in sorted EGFP negative populations using PCR. NC: genomic DNA from untransfected HEK293FTEGFP cells was used as negative control. US: genomic DNA from HEK293FTEGFP cells transfect paired Cas9 and ssODN or plasmid donor was used for PCR. S: genomic DNA from sorted EGFP negative cells from HEK293FTEGFP cells transfected with paired Cas9 and ssODN or plasmid donor