Fig. 1

CRISPR/Cas9-mediated Y chromosome elimination in vitro. a Targeted gene loci in the Y chromosome: Rbmy1a1, clustered in the short arm; Ssty2,scattered in the long arm; Kdm5d, control gene. b Experimental design. Mouse embryonic stem cells (mESCs) of XY genotype were transfected with plasmids expressing Cas9, Y chromosome-targeting sgRNAs, and mCherry. One day later, mCherry-positive mESCs were sorted by FACS and cultured in six wells for DNA-FISH analysis or 96 wells for single-cell cloning and genotyping. c Stacked bar graphs showing results of DNA-FISH analysis on the gene-edited mESCs. Percentages of cells (including dividing cell, Y:X = 2:2 or 0:2) exhibiting different genotype ratios. WT are wild-type, untransfected cells; n is the sample size of counted cells. d Representative DNA-FISH analysis of mixed ESCs targeted at Rbmy1a1, Ssty2, or Kdm5d. 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 indicate Y; red arrows indicate X. Numbered squares indicate single cells shown at a higher resolution in the right panels. Bar, 20 μm. e Genotyping analysis of ESC clones from Rbmy1a1 and Ssty2 targeting. Controls: Kdm5d targetted and untransfected (WT) cells. Sry and Mecp2 are located on Y and X, respectively. f Efficiency of Y chromosome elimination by Rbmy1a1 and Ssty2 targeting. The experimental groups (Rbmy1a1 and Ssty2) showed more Sry-negative clones than the control group (Kdm5d and WT; ***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). g Karyotyping of Sry-negative ESCs showed 39 instead of 40 chromosomes. Bar, 20 μm. h Representative DNA-FISH analysis of single ESC clones targeted at either Rbmy1a1 or Ssty2. Bar, 20 μm. i Whole genome sequencing showing Y chromosome elimination of XO ESCs. The XO ESCs (Rbmy1a1, #2; Ssty2, #3) showed one copy of X with Y absent. WT, untreated mouse. Vertical axis, copy number; horizontal axis, chromosome number