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, 18 (1), 224

CRISPR/Cas9-mediated Targeted Chromosome Elimination

Affiliations

CRISPR/Cas9-mediated Targeted Chromosome Elimination

Erwei Zuo et al. Genome Biol.

Abstract

Background: The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown.

Results: Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells.

Conclusions: CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.

Conflict of interest statement

Ethics approval and consent to participate

The use and care of animals complied with the guidelines of the Biomedical Research Ethics Committee at the Shanghai Institutes for Biological Science (CAS).

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
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
Fig. 2
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
Fig. 3
Fig. 3
Generation of mouse model with Turner syndrome by Y chromosome elimination. a Birth rate of gene-edited embryos for Y chromosome elimination. The experimental group (Rbmy1a1, Ssty1, Ssty2) showed no significant difference compared to the control group (Kdm5d). n is the sample size of transferred embryos. b Sex ratio of mice generated by gene editing. The experimental group (Rbmy1a1, Ssty1, Ssty2) showed more mice with female gonads than the control group (Kdm5d or dCas9 and sgRNA targeting Ssty2). n is the sample size of mice generated by gene editing (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). c Percentage of female mice with different karyotypes. n is the sample size of female mice. d Representative image of the XO karyotype of a female generated by Ssty2 targeting. e Representative DNA-FISH images of cultured tail fibroblasts derived from female mice with Rbmy1a1 targeting, showing XO and XO/XY genotypes. Untreated (WT), male or female mice without gene editing. Green, Y probe; red, X probe for XqC3; blue, DNA. Green arrows, Y; red arrows, X. Numbered squares, single cells shown at a higher resolution in the right panel. Bar, 20 μm. f Results of DNA-FISH analysis on the gene-edited female mice. Percentages of cells (including dividing cells, Y:X = 2:2 or 0:2) exhibiting different genotype ratios. Data include XO mice (Rbmy1a1, mice 2 to 5; Ssty1, mice 1 to 4; Ssty2, mice 1 to 5; see Additional file 1: Table S3 for corresponding mice) as well as mosaic XO/XY mice (Rbmy1a1, mice 1, 3, and 4). n is the sample size of counted cells
Fig. 4
Fig. 4
Identification of a mouse model with Turner syndrome generated by Y chromosome elimination. ac Rbmy1a1 (a), Ssty1 (b), Ssty2 (c) targeted mice used for genotyping (listed in Additional file 1: Table S3). The XO pure mice showed no Y chromosome-specific genes, suggesting complete elimination of the Y chromosome. Y chromosome short arm, Uba1y, Kdm5d, Eifs23y, Dxd3y, Usp9y, Tspy, Sry, Rbmy1a1; Y chromosome long arm, Ssty1, Ssty2. Gapdh, control gene in autosome. d WGS showed Y chromosome elimination of XO mice. Histograms of X and Y chromosome are shown in the right panel at a higher resolution. The XO mice (Rbmy1a1, #2 and Ssty1, #1) showed one copy of the X chromosome with the Y chromosome absent. Vertical axis, copy number; horizontal axis, chromosome number. e Twelve-week old XO mouse from Ssty2 XO #4 showing normal female genitals (green arrowheads) and nipples (red arrowheads). WT, female mice without gene editing. f, g Fertility of mice with Y chromosome deletion. The gene-edited XO female mice (number indicated above) were paired with wild-type male mice for over 3 months. The frequency of pregnancy (f) and litter size (g) was determined and compared with those of XX siblings (**P < 0.01, *P < 0.05, not significant (n.s.), P > 0.05, Chi-square test). h The weight of XO mice (Rbmy1a1, Ssty1, Ssty2) and the siblings of XX mice. The mice were measured about once a week from 1 week to 9 weeks. Means ± SEM
Fig. 5
Fig. 5
Elimination of mouse Y chromosome in zygotes with an sgRNA cocktail. a Schema of targeted gene loci in Y chromosome. 7 one-copy genes, each targeted with two sgRNAs, are in the short arm. b, c The blastocyst (b) and birth rate (c) of embryos with 14-sgRNA cocktail for Y chromosome elimination. Control: Kdm5d. ‘n’: sample size. d Sex ratio of the mice generated by gene editing. Experimental group (14-sgRNA cocktail) showed more female mice than control group (Kdm5d). (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). e Karyological characteristics of the female mice. f Genotyping analysis of the female mice obtained by 14-sgRNA targeting. The mice used for genotyping were listed in Additional file 1: Table S3. The mosaic mouse of 14-sgRNA #2 showed the integrity for the remaining Y chromosome. In contrast, the mosaic mouse of 14-sgRNA #5 showed the large deletion of short arm in the remaining Y chromosome. The XO pure mice showed no Y chromosome-specific genes, suggesting complete elimination of whole Y chromosome. g Representative DNA-FISH analysis of the female from 14-sgRNA cocktail targeting. The pure XO mouse of 14-sgRNA-XO #3 showed the absence of Y chromosome. The mosaic mouse of 14-sgRNA-XY/XO #1 showed co-existing XO and XY genotype in the cells from the same mouse. Green: a whole Y chromosome probe; red: X; blue: DNA. Arrows: Y or X chromosome. Numbered squares: single bone marrow cells shown at a higher resolution on the right or down panel. Bar, 20 μm. h Stacked bar graphs of data from DNA-FISH analysis of gene-edited female mice. Percentages of cells (including dividing cells, Y:X = 2:2 or 0:2) exhibiting different ratio of genotype. Experimental group: 14-sgRNA-XO #1 to #3; 14-sgRNA-XY/XO #1, #2. Control male mice: (WT-XY #4); control female mice: (Ssty1-XX #14, #17, Ssty2-XX #8). ‘n’: sample size of counted cells
Fig. 6
Fig. 6
Generation of mouse model with Turner syndrome by X chromosome elimination. a Targeted loci in the X chromosome. Five sgRNA target sequences (from X-A to X-E) are X chromosome-specific repeated sequences in non-coding regions. Texas red-labeled probe XqA7.3 is near the centromere. b Blastocyst rate of embryos generated by gene editing for X chromosome elimination. Embryos edited using triple sgRNAs (X-A + B + C or X-C + D + E) showed embryonic lethality, whereas those edited using a single sgRNA (X-A, X-B, X-C, or X-E) reached the blastocyst stage, with a lower blastocyst rate than the control group (Tyr-A or Tyr-A + B + C). Tyr-A or Tyr-A + B + C, a single sgRNA or triple sgRNAs targeting the locus of Tyr, a coat color gene with a single copy on the autosome. n is the sample size of cultured embryos (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). c Birth rate of embryos gene-edited for X chromosome elimination. The experimental group (X-B, X-C) showed a lower birth rate than the control group (Tyr-A). n is the sample size of transferred embryos (***P < 0.001, **P < 0.01, *P < 0.05, Chi-square test). d Percentage of female mice (X-B, X-C) with different karyotypes. Percentages of cells (including dividing cells, X = 4) exhibiting different genotype ratios. n is the sample size of female mice. e Representative image for XO karyotype of a female generated by X-C targeting. f Representative DNA-FISH analysis of the XO mice from X-B and X-C targeting. g WGS showing X chromosome elimination of XO mice. The histograms of chromosome 1 and X are shown in the right panel at a higher resolution. The XO mice (X-B, mice 1 and 3; X-C, mice 5 and 7) showed one copy of the X chromosome. Vertical axis, copy number; horizontal axis, chromosome number. h The targeted loci of XO mice (X-C, mice 4, 6, and 7) were PCR amplified and pMD-19 T TA cloned for sequencing. Indels occurred in two of three XO mice. i WGS mapping revealed that most copies of clustered repeat sequences by X-B or X-C targeting were deleted in XO mice. For X-B targeting, 72 copies of target sequences were detected in WT mice. By contrast, only one copy and two copies of target sequence were detected with mutations in X-B XO #1 and X-B XO #3, respectively. For X-C targeting, 66 copies of target sequences were detected in WT mice (untreated mice), but only two and three copies of target sequences were detected with mutations in X-C XO #5 and X-C XO #7, respectively
Fig. 7
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)
Fig. 8
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)
Fig. 9
Fig. 9
Off-target analysis in cells and animals with chromosome elimination. a Summary of indels detected by WGS. See Additional file 1: Table S3 for corresponding mice and cell lines. b Summary of genomic rearrangements on the target sequences detected by WGS. c Circos plots showing identified off-target hotspots. Lines link bait site (Kdm5d) to identified off-target hotspots of indicated CRISPR/Cas9 with a range of color from white to dark red. The higher the frequency, the darker the color. The number of identified off-targets for each CRISPR/Cas9 is shown in the middle of the circus plot. Arrowheads indicate the bait CRISPR/Cas9 Kdm5d site, but note that the bait CRISPR/Cas9 Kdm5d has no detected off-target sites. Three repeats for each treatment (Additional file 1: Table S6 and S7)
Fig. 10
Fig. 10
Mechanism of CRISPR/Cas9-mediated targeted chromosome elimination. a Results of DNA-FISH analysis on the gene-edited ES cells. Percentages of cells (including dividing cell, Y:X = 2:2 or 0:2) exhibiting different genotype ratios. n is the sample size of counted cells. b Sex ratio of mice generated by gene editing. n is the sample size of mice generated by gene editing. c Results of DNA-FISH analysis of the ratio of blastomeres with Y deletion in each male embryo with Ssty2 targeting at different stages. Data are presented as means ± SEM (n = 4 to 12; ***P < 0.001, **P < 0.01, *P < 0.05, t test). d Results of DNA-FISH analysis on cells treated with KU-55933 or not. Percentages of cells (including dividing cells, Y:X = 2:2 or 0:2) exhibiting different genotype ratios. n is the sample size of counted cells. e Examples of Y chromosome exclusion from nucleus after gene editing. DNA-FISH analysis of mouse ES cells or two-cell embryos with isolated Y chromosome following Ssty2 targeting. Green, Y; red, X; blue, DNA; N, primary nucleus; Arrows: green arrows, Y; red arrows, X. Bar, 20 μm

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