Fig. 5

Elimination of mouse Y chromosome in zygotes with an sgRNA cocktail. a Schema of targeted gene loci in Y chromosome. 7 one-copy genes, each targeted with two sgRNAs, are in the short arm. b, c The blastocyst (b) and birth rate (c) of embryos with 14-sgRNA cocktail for Y chromosome elimination. Control: Kdm5d. ‘n’: sample size. d Sex ratio of the mice generated by gene editing. Experimental group (14-sgRNA cocktail) showed more female mice than control group (Kdm5d). (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). e Karyological characteristics of the female mice. f Genotyping analysis of the female mice obtained by 14-sgRNA targeting. The mice used for genotyping were listed in Additional file 1: Table S3. The mosaic mouse of 14-sgRNA #2 showed the integrity for the remaining Y chromosome. In contrast, the mosaic mouse of 14-sgRNA #5 showed the large deletion of short arm in the remaining Y chromosome. The XO pure mice showed no Y chromosome-specific genes, suggesting complete elimination of whole Y chromosome. g Representative DNA-FISH analysis of the female from 14-sgRNA cocktail targeting. The pure XO mouse of 14-sgRNA-XO #3 showed the absence of Y chromosome. The mosaic mouse of 14-sgRNA-XY/XO #1 showed co-existing XO and XY genotype in the cells from the same mouse. Green: a whole Y chromosome probe; red: X; blue: DNA. Arrows: Y or X chromosome. Numbered squares: single bone marrow cells shown at a higher resolution on the right or down panel. Bar, 20 μm. h Stacked bar graphs of data from DNA-FISH analysis of gene-edited female mice. Percentages of cells (including dividing cells, Y:X = 2:2 or 0:2) exhibiting different ratio of genotype. Experimental group: 14-sgRNA-XO #1 to #3; 14-sgRNA-XY/XO #1, #2. Control male mice: (WT-XY #4); control female mice: (Ssty1-XX #14, #17, Ssty2-XX #8). ‘n’: sample size of counted cells