Mutation of Cdx2 in selected blastomeres in mouse early embryos using inducible nCas9ERT2. (A) Experimental procedure. Different combinations (groups C1–C3 and M) of indicated reagents were injected into one blastomere of 2-cell stage embryos. (B) Mutation ratios of Cdx2 alleles in mCherry⁺ cells of Group M or Group C3 embryos at the 8-cell stage. A Cdx2 region, including gRNA target sites, was amplified from pooled cells and cloned for sequencing. The number of sequenced clones was indicated. Mut, mutant alleles; WT, WT allele. (C) Ratios of cells with specific Cdx2 genotypes in group M embryos. The mCherry+ cells were isolated and genotyped individually. −/−, two mutant alleles; +/−, one mutant allele; WT, two WT alleles. Nc, the number of genotyped cells. (D) Structure of WT and mutant alleles as revealed in C. E1–E3, exons; gray, UTRs; arrows, direction of gRNA recognition sites; orange line, deleted region. (E and F) Representative immunofluorescent images of Cdx2 and mCherry expression (E) and ratio of cells with or without indicated markers (F) in different groups of blastocysts at E4.5, shown as (mean ± SD). (G and H) Representative immunofluorescent images of Cdx2, Nanog, and mCherry expression (G) and ratio of cells with Cdx2 or Nanog expression (H) in group M or C3 blastocysts at E4.5, shown as (mean ± SD). In F and H, the denominator was the total number of cells in individual blastocysts; P values of one-way ANOVA in the left of (F) and t test (unpaired, two-sided) in other boxplots in F and H were indicated; Ne, the number of blastocysts. Source data are available for this figure: SourceData F6.