Fig. 8

Elimination of human chromosome 21 in mouse aneuploid cells and DS iPS cells by CRISPR/Cas9-mediated gene editing. a Targeted loci in hChr21. Two sgRNA target sequences (21-A and 21-B) are specific for hChr21 repeated sequences in the non-coding regions. Control sgRNAs (21-NC-A and 21-NC-B) are hChr21-specific sequences with a unique target site. b Experimental design. Aneuploid cells were transfected with plasmids expressing Cas9, chromosome-targeting sgRNAs, and GFP. One day later, GFP-positive ES cells were sorted by FACS and cultured in six wells for DNA-FISH analysis. c Representative DNA-FISH analysis of mixed mouse ES cells targeted at either 21-A + B or control sgRNAs. Red, Texas red-labeled whole-chromosome probe for chromosome 21; green, FITC-labeled mouse chromosome 18qA1 probe; blue, Hoechst 33342-labeled DNA. Green arrows, mChr18; red arrow, hChr21. Numbered squares, single cells shown at higher resolution in the right panels. Bar, 20 μm. d Results of DNA-FISH analysis on TcH21 cells targeted at either 21-A + B or control sgRNAs (two sgRNAs with one targeting site on hChr21). Percentages of cells (including dividing cells) exhibiting different mChr18: hChr21 ratios. n is the sample size of counted cells. e Efficiency of hChr21 elimination based on DNA-FISH analysis after TcH21 cells targeted at either 21-A + B or control sgRNAs (n = 3, **P < 0.01, t test). f Representative DNA-FISH analysis of human iPSCs with trisomy 21 targeted at either 21-A + B or control sgRNAs. Green, FITC-labeled hCHr21 CEN probe; blue, Hoechst 33342-labeled DNA. Arrows, hChr21. Bar, 20 μm. g Results of DNA-FISH analysis on TcH21 cells targeted at either 21-A + B or control sgRNAs (two sgRNAs with one targeting site on hChr21). Percentages of cells (including dividing cells) exhibiting different mChr18: hChr21 ratios. n is the sample size of counted cells. h Efficiency of hChr21 elimination based on DNA-FISH analysis after DS iPSCs targeted at either 21-A + B or control sgRNAs (n = 2, **P < 0.01, Chi-square test)