Fig. 5

Comparison of homology-independent integration between dsDNA and ssDNA donors. a Schematic for evaluating homology-independent integration of mNG donors into Cas nuclease-induced DSBs. Since the Cas12a cleavage site is located inside the coding region of the TOMM20 gene, homology-independent integration of mNG into the cleavage site in the correct orientation and a correct reading frame leads to the expression of TOMM20-mNG proteins. b Flow cytometric analysis of the homology-independent integration experiment using RPE1 cells. Sense strands were used for T7 and T7RE donors. Cells at 9 days after electroporation were analyzed. Data from three biological replicates are shown. > 5,000 cells were analyzed for each sample. c Representative images from the homology-independent integration experiment. Cells at 11 days after electroporation were fixed and analyzed. Scale bar: 10 µm. d Schematic overview of the workflow for evaluating homology-independent integration of GALNT2-mNG cassettes into Cas12a nuclease-induced DSBs. Non-integrated cassettes are cleared from cells during long-term culture for more than 10 days. The cassettes inserted into the genome produce doxycycline (dox)-induced expression of the Golgi protein GALNT2-mNG. e Flow cytometric analysis of the cassette integration experiment using RPE1 cells. Cells at 20 days after electroporation were treated with 1 µg/mL of doxycycline (dox) for 24 h and analyzed. Data from three biological replicates are shown. Approximately 20,000 cells were analyzed for each sample. f Representative images from the cassette integration experiment. Cells at 13 days after electroporation were treated with 1 µg/mL of doxycycline (dox) for 24 h and fixed for analysis. Arrowheads indicate cells with Golgi-like mNG signals. Scale bar: 100 µm (the left panel), 10 µm (the right panel). Data are presented as mean ± S.D. A two-tailed, unpaired Student’s t-test was used to obtain the P-value. n.s.: Not significant