Fig. 1

An optimized knock-in method using long dsDNA donors for efficient endogenous tagging with fluorescent proteins. a Schematic overview of long-dsDNA-based endogenous gene tagging in human RPE1 cells. b Representative images of RPE1 cells with Cas12a-mediated endogenous mNG tagging of the indicated genes. Cells at 7–12 days after electroporation were fixed and analyzed. Scale bar: 10 µm. c, Representative images of RPE1 cells with Cas9-mediated endogenous mNG tagging of the indicated genes. Cells at 12–17 days after electroporation were fixed and analyzed. Scale bar: 10 µm. d Genomic PCR detecting the mNG insertion into the indicated genes with Cas12a or Cas9-mediated knock-in in RPE1 cells. The primers were designed to amplify the 5’ junction of the mNG insertion for each gene. TUBB5: loading control. LHA: left HA, RHA: right HA. e Western blotting confirming the fusion of mNG to HNRNPA1 in RPE1 cells via the Cas12a-mediated knock-in method. The FACS-enriched, mNG-positive knock-in cells were used. HSP90: loading control. WB: Western blotting. f Flow cytometric analysis of Cas12a-mediated HNRNPA1-mNG and TOMM20-mNG knock-in RPE1 cells. Cells at 8 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. g Quantification of percentages of mNG-positive cells from (f). Data from three biological replicates are shown. > 5,000 cells were analyzed for each sample of HNRNPA1 and TOMM20. Data are represented as mean ± S.D. h Flow cytometric analysis of Cas9-mediated HNRNPA1-mNG knock-in RPE1 cells. The donor concentration was 33 nM. Cells at 5 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. i Quantification of percentages of mNG-positive cells from (h). Two different concentrations of the dsDNA donor were analyzed. Data from three biological replicates are shown. 10,000 cells were analyzed for each sample. Data are represented as mean ± S.D. Full-length blots and gels are presented in Fig. S6