Fig. 6

Generation of mouse model with Turner syndrome by X chromosome elimination. a Targeted loci in the X chromosome. Five sgRNA target sequences (from X-A to X-E) are X chromosome-specific repeated sequences in non-coding regions. Texas red-labeled probe XqA7.3 is near the centromere. b Blastocyst rate of embryos generated by gene editing for X chromosome elimination. Embryos edited using triple sgRNAs (X-A + B + C or X-C + D + E) showed embryonic lethality, whereas those edited using a single sgRNA (X-A, X-B, X-C, or X-E) reached the blastocyst stage, with a lower blastocyst rate than the control group (Tyr-A or Tyr-A + B + C). Tyr-A or Tyr-A + B + C, a single sgRNA or triple sgRNAs targeting the locus of Tyr, a coat color gene with a single copy on the autosome. n is the sample size of cultured embryos (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). c Birth rate of embryos gene-edited for X chromosome elimination. The experimental group (X-B, X-C) showed a lower birth rate than the control group (Tyr-A). n is the sample size of transferred embryos (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). d Percentage of female mice (X-B, X-C) with different karyotypes. Percentages of cells (including dividing cells, X = 4) exhibiting different genotype ratios. n is the sample size of female mice. e Representative image for XO karyotype of a female generated by X-C targeting. f Representative DNA-FISH analysis of the XO mice from X-B and X-C targeting. g WGS showing X chromosome elimination of XO mice. The histograms of chromosome 1 and X are shown in the right panel at a higher resolution. The XO mice (X-B, mice 1 and 3; X-C, mice 5 and 7) showed one copy of the X chromosome. Vertical axis, copy number; horizontal axis, chromosome number. h The targeted loci of XO mice (X-C, mice 4, 6, and 7) were PCR amplified and pMD-19 T TA cloned for sequencing. Indels occurred in two of three XO mice. i WGS mapping revealed that most copies of clustered repeat sequences by X-B or X-C targeting were deleted in XO mice. For X-B targeting, 72 copies of target sequences were detected in WT mice. By contrast, only one copy and two copies of target sequence were detected with mutations in X-B XO #1 and X-B XO #3, respectively. For X-C targeting, 66 copies of target sequences were detected in WT mice (untreated mice), but only two and three copies of target sequences were detected with mutations in X-C XO #5 and X-C XO #7, respectively