Fig. 2

Elimination of the Y chromosome in zygotes by CRISPR/Cas9-mediated gene editing. a Targeted gene loci in the Y chromosome: Rbmy1a1, clustered in the short arm; Ssty1, Ssty2, scattered in the long arm; Kdm5d, control gene. b Experimental design. Cas9 mRNA and two specific sgRNAs that targeted the Rmby1a1, Ssty1, Ssty2, or Kdm5d locus were injected into individual mouse zygotes, which were further cultured to 4- to 16-cell embryos for DNA-FISH analysis or transferred into recipients. WT, embryos without injection of Cas9 mixture. c Blastocyst rate of embryos generated by gene editing. The experimental group (Rbmy1a1, Ssty1, Ssty2) showed no difference compared to the control group (Kdm5d or WT); n is the sample size of injected embryos. d Representative DNA-FISH analysis of 4- to 16-cell embryos after Rbmy1a1 targeting. Green, FITC-labeled whole-chromosome probe for Y chromosome; red, Texas red-labeled X chromosome probe for XqA7.3; blue, Hoechst 33342-labeled DNA. Green arrows, Y; red arrows, X. WT male embryo (XY), one green signal and one red dot; WT female embryo (XX), two red dots, no green signal; pure XO embryo (XO), one red dot, no green signal; mosaic embryo (XY/XO), co-existing XO and XY genotype in the blastomeres from the same embryo. Insets: single blastomeres shown at higher resolution. Bar, 50 μm. e, f Results of DNA-FISH analysis of the ratio of blastomeres with Y deletion (e) and sex chromosomal genotype (f) in each male embryo at 4- to 16-cell stage. Experimental group, Rbmy1a1, Ssty1, Ssty2; control group, Kdm5d and WT (***P < 0.001, not significant > 0.05, t test). n represents the sample size of blastomeres in e and sample size of embryos in f. Error bars represent SEM