Fig. 1

Targeted elimination of human Y chromosome. A gRNAs targeted sites on human Y chromosome are indicated. B Experimental plan: a mixture of pSpCas9(BB)-2A-GFP vectors containing gRNA sequence for TSPY1, RBMY1A1 and DYZ3 was used to transfect ARPE-19. Cells were treated for 16 h after transfection with the ATM inhibitor KU-55933. After subcloning, single clones have been picked, expanded, and then screened by FISH. The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. C DNA-FISH analysis on interphase and metaphase (D) spreads indicating the complete loss of Y chromosome. Green whole chromosome paint (WCP) probe was used for Y chromosome hybridization. Red WCP probe was used for X chromosome identification. E Agarose gel electrophoresis of RBMY1A1, SRY and TSPY1 PCR products derived from ARPE-19 Y-negative clone 3. GAPDH has been used as reference control gene. F Karyotype analyses of ARPE-19 and Y-negative clone 3. Red arrows indicate chromosomal alterations in ARPE-19 cells. Green arrow shows Y chromosome elimination in Y-depleted clone 3