Nuclear translocation and gene editing effectiveness of nCas9ERT2. (A) 4-OHT–induced nuclear translocation of nCas9^ERT2 in human HEK293T cells. Cells were transfected with an expression construct (top) and immunostained with Cas9 antibody and DAPI staining (bottom). The images shown were representative images with Cas9-positive cells outlined by the dashed line. N1, SV40 NLS; N2, nucleoplasmin NLS. (B) Gene editing effectiveness of 4-OHT–induced nCas9^ERT2 in HEK293T cells. Endogenous EMX1 was the target. Top, compositions of constructs with designated numbers for transfection (T, T2A); bottom, gel electrophoretic image showing T7E1 assay outcome of EMX1 locus. The mutant band was indicated by arrows. (C) The indicated transgenic embryos express mCherry specifically in PGCs (indicated by arrowheads), confirming the PGC specificity of the zebrafish piwil1 promoter. Inserts showed arrowhead-pointed areas. (D) Expression pattern of nCas9^ERT2 in indicated transgenic embryos at different stages. Embryos were immunostained with Cas9 and Ddx4 (a PGC-specific marker) antibodies plus DAPI staining. Note that Cas9 started to be specifically expressed in PGCs from the sphere stage onward. Inserts showed enlarged areas harboring PGCs. (E) Bar plot showing the ratios of Cas9-positive PGCs (Ddx4 and Cas9 double-positive cells) to total PGCs (Ddx4-positive cells) in Tg(piwil1:nCas9^ERT2-n3U) embryos we analyzed in at least three replicates. No. PGCs, the number of PGCs at each stage. Source data are available for this figure: SourceData F1.