Fig. 2

Targeted integration of a single donor plasmid into the CCR5 locus in HeLa cells through single crossover. a Schematic diagram of forward integration of the EGFP donor plasmid into the human genome through the generation of double-strand breaks at the target sites of CRISPR/Cas9 or ZFN in the genome and plasmids via single crossover or NHEJ. Two homology arms flanking the target sites of the engineered nucleases are shown in blue and yellow. vLHA and gLHA represent the left homology arms on the vector and genome, respectively; vRHA and gRHA represent the right homology arms on the vector and genome, respectively. b Schematic diagram of reverse integration of the EGFP donor plasmid into the human genome via NHEJ through the generation of double-strand breaks at the target sites of CRISPR/Cas9 or ZFN in the genome and plasmids. c Targeted knock-in of donor plasmids in the forward orientation but not in the reverse orientation by ZFN or CRISPR/Cas9 was detected by junction PCR. d DNA sequences of the wild-type (WT) and mutant clones. 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 indicated by 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. e Brightfield and fluorescence microscopy images of HeLa clonal cells. Scale bar = 50 μm. f The frequencies of targeted integration through single crossover mediated by CRISPR/Cas9 or ZFN was detected through junction PCR (represented by the 5′ junction PCR results; similar results of the 3′ junction PCR analysis are not shown)