Fig. 7
Elimination of human chromosome 14 in cells by CRISPR/Cas9-mediated gene editing. a Targeted loci in human chromosome 14. Six sgRNA target sequences (from 14-A to 14-F) are specific for human chromosome 14 repeated sequences in the non-coding regions. FITC-labeled probe 14q11.2 is near the centromere. Red arrow, insertion site of PB-CAG-mCherry; green arrows, gene loci for genotyping. b Establishment of aneuploid mouse ES cells with hChr14 (TcH14). c Experimental design. Aneuploid cells were transfected with plasmids expressing Cas9, chromosome targeting sgRNAs, and mCherry. One day later, GFP-positive ES cells were sorted by FACS and cultured in six wells for DNA-FISH analysis or 96 wells for single cell cloning and genotyping. d Percentage of mCherry-negative cells after gene editing. TcH14 cells were transfected with px330 plasmid containing different sgRNAs and then sorted by FACS 1 day later. Three days later, transfected TcH14 cells were analyzed by FACS. Data are presented as means ± SEM (n = 3, ***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). e Genotyping analysis of mCherry-negative cell lines derived from gene editing on TcH14 cells. Note that hChr14 in the cell lines (14-A + F #2, #6, #7; 14-F #3, #6) were partial deletions. f Representative DNA-FISH analysis of clones #1 and #8 from 14-A + F targeting. Green, human chromosome 14 probe for 14q11.2; red, X probe for XqA7.3; blue, DNA; green arrow, human chromosome 14; red arrows, mouse X chromosome. Numbered squares, single cells shown at higher resolution in the bottom panels. Bar, 50 μm. g Results of DNA-FISH analysis on mCherry-negative clones from 14-A + F targeting. Percentages of cells (including dividing cells, X:14 = 4:2 or 4:0) exhibiting different X:14 ratios. Data include TcH14 mCherry-negative clones (14-A + F #1 to #8) as well as control TcH14 mCherry-positive clones. n is the sample size of counted cells. h The weight of XO mice (X-B, n = 3; X-C, n = 3) and the siblings of XX mice (n = 6) was measured about once a week from 1 to 9 weeks. Means ± SEM. i RNA-seq analysis of TcH14 cells and cells with chromosome correction. The corrected cell lines (14-A + F #1 and #8) showed no gene expression on hChr14 (BMP4 for example)