Fig. 3

Comparison of knock-in efficiency between dsDNA and ssDNA long donors. a Representative images of RPE1 cells with mNG tagging to the indicated genes using T7 or T7RE donors. The indicated Cas nuclease was used for each condition. For ssDNA donors, sense strands were used. Cells at 7–13 days after electroporation were fixed and analyzed. Scale bar: 10 µm. b Flow cytometric analysis of Cas12a-mediated HNRNPA1-mNG knock-in RPE1 cells, using dsDNA, T7, and T7RE donors. Cells at 12 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. c Quantification of percentages of mNG-positive cells from (b). Data from three biological replicates are shown. Approximately 10,000 cells were analyzed for each sample. d Flow cytometric quantification of mNG-positive cells in Cas12a-mediated TOMM20-mNG knock-in RPE1 cells, using the indicated donors. Cells at 9 days after electroporation were analyzed. Data from three biological replicates are shown. > 5,000 cells were analyzed for each sample. e Flow cytometric quantification of mNG-positive cells in Cas9-mediated HNRNPA1-mNG knock-in RPE1 cells, using the indicated donors at 33 nM. Cells at 6 days after electroporation were analyzed. Data from three biological replicates are shown. Approximately 10,000 cells were analyzed for each sample. f Titration of the indicated donors for mNG tagging of HNRNPA1 using Cas12a in RPE1 cells. Cells at 11 days (dsDNA) or 10 days (T7 and T7RE) after electroporation were analyzed. For ssDNA donors, sense strands were used. Data from three biological replicates are shown. > 8,000 cells were analyzed for each sample. n.a.: Not analyzed. Data are presented as mean ± S.D. P-value was calculated by the Tukey–Kramer test. ***P < 0.001, n.s.: Not significant