Fig. 2

Establishment and application of HiHo-AID2 in A431 cells. a Scheme of genetic modifications for degron-GFP tagging through conventional procedure (control) and one-step procedure (coIN) to assess tagging efficiencies in b–e. PuroR: puromycin-resistance gene. b, c Comparison of degron-GFP tagging efficiencies in conventional procedure and one-step procedure with 1 μM M3814 as HDR enhancer as indicated. A representative FACS profile (b) and statistics of percentage of GFP-positive cells (c, left), the percentual change of single-cell GFP intensity compared to control (conventional procedure) (c, middle), and the cell count (× 10⁶ cells/ml) (c, right) are shown. N = 16 pairs of tagging plasmids. Statistical comparisons are shown in Additional file 4: Table S6. d, e Comparison of degron-GFP tagging efficiencies in one-step procedure using different HDR enhancers. A representative FACS profile (d) and graphs depicting the percentage of GFP-positive cells (e, left), the percentual change of single-cell GFP intensity to control (coIN) (e, middle), and the cell count (× 10⁶ cells/ml) (e, right) are shown. N = 10 pairs of tagging plasmids. Numbers above columns indicate mean values; lines link the same endogenous tagging pairs in c, e. Statistical comparisons are shown in Additional file 4: Table S6. f Scheme of the genomic modifications in HiHo-AID2. BSD: Blasticidin S deaminase. Psen and Pan indicate the primer set for genotyping PCR of either N- or C-terminal tagging. g Scheme of a representative genotyping PCR result showing heterozygous and homozygous tagging at endogenous loci in the AID clones. h Genotyping PCR results of SAC1 clones generated with or without i53 plus 0.25 μM M3814 as HDR enhancers. i Graphs depicting the genotyping PCR results for 8 targets without HDR enhancer, and 11 targets with either 1 μM M3814 or i53 plus 0.25 μM M3814 as HDR enhancers. Numbers above columns indicate total amount of clones analyzed. j WB analysis of inducible SAC1 degradation. k Graph showing the PI4P staining intensity upon SAC1 degradation in A431 wild-type and SAC1 AID cells. One-way ANOVA, n.s.: non-significant, ****p < 0.001. N = 17 fields. All statistical comparisons are shown in Additional file 4: Table S6. l Widefield imaging of cell morphological changes upon SAC1 degradation. Scale bar: 50 μM. Representative of 2 clones (j, k). WT: wild-type; Hetero: heterozygous; Homo: homozygous; pico: 0.5 μM pico_cvxIAA treatment; a.u. arbitrary unit