Fig. 4
The design of single-stranded oligodeoxynucleotides (ssODNs) and plasmid donor with blocking mutations, and the comparison of HDR efficiencies between TALEN and CRISPR/Cas9. a The schematic diagram shows two 120-mer ssODN and a plasmid donor DNA with blocking mutations (BMs) 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. A silent G to T mutation was introduced on ssODN1-BM and EBFP-BM plasmid to create a silent HindIII site, and a silent G to C mutation was introduced on ssODN2-BM to create a silent XhoI site. More silent mutations were introduced on ssODN and plasmid template to form the blocking mutations (BMs) for each TALEN arm and gRNA. b Representatives of the FACS analysis of EBFP positive cells generated via HDR by using TALEN or CRISPR/Cas9 with donor templates with blocking mutations. NG: EGFP negative cells. c The quantification of EBFP positive cells in extended cultured EGFP negative cells sorted from HEK293FTEGFP cells transfected with TALEN or CRISPR/Cas9 conjugated with donor templates with blocking mutations. In each case, a total of 10,000 events were counted