Fig. 3

Targeted integration of multiple donor plasmids into the CCR5 locus in HeLa cells through single crossover. a Schematic diagram of the successive integration of EGFP and DsRed donor plasmids into the CCR5 locus in HeLa cells through single crossover. b Junction PCR analysis of targeted integration. c Sequence analysis of induced mutations at target sites of the junction PCR amplicon from HeLa cells transfected with only the EGFP donor plasmid (EGFP⁺/DsRed⁻), and cells transfected with both EGFP and DsRed donor plasmids (EGFP⁺/DsRed⁺). The binding sites of the ZFN pair and sgRNA are shown in yellow and green bars, respectively. The PAM sequence is shown in red and underlined. Deleted and inserted bases are presented in dashes and blue letters, respectively. The number of inserted or deleted bases, and the ratio of the number of mutant clones to the number of total clones are indicated in the parentheses. d Brightfield and fluorescence microscopy images of clonal HeLa cells. Scale bar = 50 μm. e The frequency of targeted multiple integration through single crossover mediated by CRISPR/Cas9 or ZFN was detected through junction PCR analysis