- Open Access
CRISPR/Cas9-mediated targeted chromosome elimination
- Erwei Zuo†1,
- Xiaona Huo†1,
- Xuan Yao†1,
- Xinde Hu†1,
- Yidi Sun†3,
- Jianhang Yin†2,
- Bingbing He1, 4,
- Xing Wang1, 4,
- Linyu Shi1,
- Jie Ping5,
- Yu Wei1, 6,
- Wenqin Ying1,
- Wei Wei1, 7,
- Wenjia Liu1,
- Cheng Tang1,
- Yixue Li3,
- Jiazhi Hu2Email author and
- Hui Yang1Email author
© The Author(s). 2017
- Received: 5 July 2017
- Accepted: 26 October 2017
- Published: 24 November 2017
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.
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.
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.
Aneuploidy is a human genetic disorder due to the addition or deletion of a chromosome, leading to significant morbidity and mortality during infancy or childhood . The past decade has witnessed major advances in strategies to correct single-gene defects of rare monogenic disorders, beginning with in vitro experiments and in several cases advancing to in vivo studies and clinical trials. By contrast, only a few attempts have been made to genetically correct the over-dose of genes for an entire chromosome in aneuploid cells. Targeted chromosome elimination could be achieved by insertion of oppositely oriented loxP sites into the targeted chromosome followed by Cre-mediated sister-chromatid recombination , or by insertion of a TKNEO transgene into one copy of a targeted chromosome followed by drug selection of chromosome-deletion clones via spontaneous chromosome loss . Both of these approaches require two-step manipulation and resulted in low yields of chromosome-deleted cells, and are thus unsuitable for in vivo studies. Alternatively, over-dose of genes in aneuploid cells could be corrected by insertion of a large, inducible XIST transgene into the targeted chromosome to silence one copy of it . However, the efficiency of the targeted insertion was very low and some genes may have escaped from inactivation.
The type II bacterial CRISPR/Cas9 system has been engineered into an efficient genome-editing tool consisting of the Cas9 nuclease and a single guide RNA (sgRNA), dramatically transforming our ability to edit the genomes of diverse organisms. The sgRNA targets Cas9 to genomic regions to induce double-stranded DNA breaks, which are repaired by nonhomologous end-joining or homology-directed repair. CRISPR/Cas9-mediated genome editing has been applied to generate cells or animals carrying precise gene mutations [5, 6], including rearrangements [7, 8] and deletion of chromosome segments . We asked whether this powerful technology could be used for targeted chromosome elimination to generate animal models with chromosome deletion in various species and to treat human aneuploidy diseases involving chromosome addition.
In this study we report a novel application of CRISPR/Cas9 technology; the selective elimination of a single specific chromosome via multiple DNA cleavages on the targeted chromosome in cultured cells, embryos, and in vivo tissues. These cleavages were induced by a single sgRNA or two sgRNAs that targeted multiple chromosome-specific sites, or by a cocktail of 14 sgRNAs, with each targeting one specific site. More importantly, this approach eliminated human chromosome 21 (hChr21) in human induced pluripotent stem cells (iPSCs) with trisomy 21. CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to developing animal models and therapeutic treatments for aneuploidy.
Elimination of the Y chromosome in vitro and in vivo
To examine whether chromosome deletion could be achieved directly by CRISPR/Cas9 editing in established mouse embryonic stem (ES) cells, we designed a sgRNA that targeted the locus consisting of more than 50 repeats of an RNA-binding motif gene on the Y chromosome (Rbmy1a1), which are clustered in the short arm . Alternatively, we targeted the spermiogenesis-specific transcript on Y 2 (Ssty2)  that contains repeated gene sequences scattered in the long arm (Fig. 1a). One day after transfecting mouse ES cells of XY genotype with plasmids expressing Cas9, Y chromosome-targeting sgRNAs, and mCherry, we sorted mCherry-positive ES cells by FACS and cultured them on feeder cells (Fig. 1b). To detect whether the Y chromosome was eliminated, we performed DNA-FISH (fluorescence in situ hybridization) analysis 3 days later using a whole-chromosome probe for the Y chromosome and near-centromere probe XqA7.3 for the X chromosome (see “Methods”) on the transfected cells. We found that about 30 and 50% of ES cells targeted for Rbmy1a1 and Ssty2, respectively, had no Y chromosome signals, indicating Y chromosome elimination in some culture cells (Fig. 1c, d). This efficiency of chromosome deletion was much higher than that achieved via spontaneous chromosome loss or Cre-mediated chromosome deletion in previous studies (<10−4) [2, 3]. To further confirm Y chromosome elimination, single clones derived from transfected cells were randomly picked, expanded, and genotyped. We found the absence of the Y-specific gene Sry (on the short arm of the Y chromosome) in 4/18 (22%) clones with Ssty2 targeting and 10/52 (19%) clones with Rbmy1a1 targeting (Fig. 1e, f). Karyotyping of Sry-negative ES cells showed 39 instead of 40 chromosomes (Fig. 1g; Additional file 1: Table S3), and DNA FISH and whole genome sequencing (WGS) further confirmed the complete deletion of the Y chromosome (Fig. 1c, h, i).
To test whether the Y chromosome could be eliminated in vivo by CRISPR-Cas9 editing, we delivered the sgRNA-Ssty2-EGFP construct targeting the Y chromosome to E14.5 mouse brain via in utero electroporation (Additional file 1: Figure S1a). Two days after electroporation, we sorted EGFP-positive cells in the male brain by FACS and performed DNA-FISH (Additional file 1: Figure S1b). We found that about 40% of EGFP-positive cells showed no Y chromosome signal (Additional file 1: Figure S1c–e). By contrast, only 1% of wild-type (WT) cells and 8% of EGFP-negative cells in the brain contained no Y chromosome signal (Additional file 1: Figure S1c–e). These results indicate that the Y chromosome could be efficiently eliminated in vivo.
Together, these results indicate that the Y chromosome could be selectively eliminated in vitro and in vivo by CRISPR/Cas9-mediated multiple cuts at chromosome-specific repeated sequences.
Generation of a mouse model with Turner syndrome by Y chromosome elimination
Compared to the siblings with the XX karyotype, XO mice obtained by our gene-editing approach showed normal body weight (Fig. 4e, h; Additional file 1: Table S4), consistent with previous reports [12–14]. However, XO mice or XO/XY mosaic mice from an inbred C57BL/6 background showed reproductive defects compared to their wild-type counterparts, including the frequency of pregnancy and parturition (Fig. 4f, g; Additional file 1: Table S4), all of which are found in patients with Turner syndrome [15, 16]. Interestingly, many patients with aneuploidy diseases (e.g., Turner syndrome) often show mosaicism . Our approach is an efficient way to generate aneuploidy mouse models with mosaicism, which is not found in previous models .
Generation of mouse model with Turner Syndrome by X chromosome elimination
In addition to establishing a chromosome-deleted mouse model, we have also derived ES cells from blastocysts that were gene-edited by single sgRNA for X chromosome elimination. We obtained 25 out of 52 ES cell lines without a Y chromosome, and examined 10 out of 25 female ES cell lines for further karyotyping. We found that two of them showed a pure XO karyotype, as confirmed by DNA-FISH (Additional file 1: Figure S2b, d). These ES cell lines with distinct karyotypes could be useful for studying the effect of chromosome deletion at the cellular level.
CRISPR/Cas9-mediated gene editing promotes autosome elimination
Taken together, these results indicate that multiple CRISPR/Cas9-induced DNA cleavages could promote targeted autosome loss in aneuploid ES cells, as well as cancer cells.
Off-target effects of CRISPR/Cas9-mediated chromosome elimination
Given that the sample sizes of examined cell clones or mice by WGS and FISH were small and the results cannot reveal rare off-target effects in a population of cells or organisms, we employed high-throughput genome-wide translocation sequencing (HTGTS)  to assess off-target activities of the CRISRP/Cas9 systems. To improve the sensitivity of HTGTS, we introduced magnetic bead-mediated DNA recovery after linear amplification to remove extra biotinylated primers and the final libraries were subjected to Hiseq sequencing (see “Methods” for details). Using CRISRP/Cas9 cutting sites at the Kdm5d locus as the improved HTGTS (iHTGTS) bait, we barely detected any off-target hotspots for CRISRP/Cas9 targeting the Kdm5d site, but did identify several off-target sites for CRISRP/Cas9 targeting the Ssty1 or Ssty2 locus (Fig. 9c; Additional file 1: Tables S6 and S7). The off-target sites located in autosomes were identical to those from the WGS results. Additionally, the majority of the determined off-target hotspots were located in the Y chromosome, which was invisible to WGS and FISH in Y chromosome-deleted cells. These Y chromosome-containing off-target sites might further promote Y deletion during chromosome elimination mediated by CRISRP/Cas9, but note that the strongest hotspots harbored only one or two mutation sites in the Cas9-recognition sequences, which should be easily located by bioinformatic prediction. Therefore, strong off-target sites, especially the ones in the same chromosome, should be taken into account during chromosome-elimination using CRISRP/Cas9.
Together, these results indicate that CRISPR/Cas9-mediated chromosome elimination did not induce significant off-target alteration in chromosome-deleted mice and cell lines beyond that expected for CRISPR/Cas9-mediated editing in general [5, 25, 27, 28].
Mechanism of CRISPR/Cas9-mediated targeted chromosome elimination
We next examined whether multiple CRISPR/Cas9-mediated cleavages on a targeted chromosome produce micronuclei, resulting from pulverization of chromosomes . After cells were treated with Ssty2 sgRNA, we found that micronuclei-containing Y chromosome was observed around the primary nucleus of cells (Fig. 10e). This chromosome loss may be caused by nuclear exclusion of the targeted chromosome followed by cytoplasmic degradation, a process that requires further study.
A very recent study  reported that the Y chromosome could be deleted in ES cells and zygotes by CRISPR/Cas9-mediated genome editing. Here we have achieved complete elimination of the Y chromosome by multiple CRISPR/Cas9-mediated DNA cleavages on the targeted chromosome in ES cells, cells in vivo, and zygotes with high efficiency. Notably, using this approach to eliminate the X chromosome in mouse embryos with the XX karyotype, one of two homologous X chromosomes could be efficiently deleted. However, the remaining X chromosome was also mutated, with indels or fragment deletion in the targeted region. Most chromosome-specific repeated sequences are located in non-coding regions, and thus we could minimize these side effects by targeting the non-coding DNA sequences within small regions (< 2 kb) without obvious biological functions. Alternatively, in principle, these indels and large deletions could be avoided by sgRNAs that target only one of the two homologous chromosomes, based on single nucleotide polymorphism. As shown in Fig. 5, we could delete the Y chromosome with 14 single-target sgRNAs. Nevertheless, reducing the number of sgRNAs and improving the efficiency of chromosome elimination may make this approach more applicable.
We have shown that multiple CRISPR/Cas9-induced DNA cleavages could promote extra hChr14 or hChr21 loss in aneuploid ES cells, as well as hChr7 in human cancer cells. However, we failed to obtain aneuploid mouse ES cells or embryos with autosome deletion (data not shown). We surmise that single-autosome deletion would inhibit cell growth or lead to embryonic lethality. Thus, using aneuploid cells with trisomy autosomes, such as cells from DS patients containing three hChr21s, we detected autosome elimination by CRISPR/Cas9 editing.
Recently, Yang et al.  reported that CRISPR/Cas9-mediated editing of porcine endogenous retroviruses (PERVs) could remove repetitive sequences (up to 62 copies) but did not delete chromosomes. In comparison with this study, our strategy to eliminate chromosomes is to use sgRNAs targeting repeat sequences in a single chromosome, rather than repeat sequences scattered in many chromosomes. Consistent with this study , we observed no obvious off-target mutations or chromosome rearrangements in all examined mouse ES cells and mice with chromosome elimination. Notably, we observed that multiple CRISRP/Cas9-mediated DNA cleavages could produce partial deletion of the targeted chromosome in mice, aneuploid mouse ES cells, and cancer cells, as well as chromosome rearrangement in cancer cells. Furthermore, several off-target sites for CRISRP/Cas9 targeting the Ssty1 or Ssty2 locus were detected by both genome-wide off-target assay (iHTGTS) and independent WGS analysis. Therefore, the evaluation of off-target effects by both in silico and in vivo approaches should be taken into account when designing CRISRP/Cas9 systems for chromosome elimination and may be needed before this approach can be used clinically without risk.
Although there are many mouse models for aneuploidy diseases, such as the XO mouse for Turner syndrome, many features of patients with Turner syndrome could not be well replicated in those mouse models, including the two most common—short stature and premature ovarian failure—which affect over 90% of recognized patients [15, 18]. CRISPR/Cas9-mediated targeted chromosome elimination dramatically transforms our ability to generate disease models in diverse organisms, such as in non-human primates. Moreover, this approach would provide a potential therapeutic approach to cure aneuploidy diseases, including DS, Klinefelter syndrome, and XYY syndrome [3, 4, 21, 32–36].
Aneuploidy is a hallmark of cancer , and although it can impair cell proliferation and change cell metabolism, it could also promote cell growth under selective pressure, in which context it might contribute to tumorigenesis [38, 39]. Compounds such as AICAR, chloroquine, and 17-AAG, which cause lethality only in aneuploid cells, are in clinical trials of their antitumor activity in multiple myeloma and anaplastic large cell lymphoma . CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach for studying aneuploidy in tumorigenesis and a potential treatment strategy against a broad spectrum of human tumors.
Production of Cas9 mRNA and sgRNA
Bicistronic expression vector px330 expressing Cas9, mCherry, and sgRNA was digested with BbsI, and the linearized vector was purified using the Universal DNA Purification Kit (Tiangen). A pair of oligos (Additional file 1: Table S8) for each targeting site was annealed, phosphorylated, and ligated to the linearized vector. The T7 promoter was added to the Cas9 coding region by PCR amplification of px260, using primer Cas9 F and R (Additional file 1: Table S8). The T7-Cas9 PCR product was purified using the Universal DNA Purification Kit (Tiangen) and used as the template for in vitro transcription (IVT) using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). The T7 promoter was added to the sgRNA template by PCR amplification of px330, using primers listed in Additional file 1: Table S8. The T7-sgRNA PCR product was purified and used as the template for IVT using a MEGA shortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the sgRNAs were purified using a MEGAclear kit (Life Technologies) and eluted in RNase-free water.
One-cell embryo injection
All animal procedures were performed under the ethical guidelines of the Institute of Neuroscience, Chinese Academy of Sciences. C57BL/6 J or B6D2F1 female mice and ICR mouse strains were used as embryo donors and foster mothers, respectively. Super-ovulated female C57BL/6 J (4 weeks old) or B6D2F1 mice (7–8 weeks old) were mated to C57BL/6 J or B6D2F1 stud males, and fertilized embryos were collected from the oviduct. Cas9 mRNA (50 ng/μl) and sgRNA (50 ng/μl for each sgRNA in single to three sgRNA injections, 20 ng/μl for each sgRNA in the 14-sgRNA cocktail injection) were injected into the cytoplasm of fertilized eggs with well-recognized pronuclei in HCZB medium containing 10 μg/ml Cytochalasin B (CB). The injected zygotes were cultured in KSOM with amino acids at 37 °C under 5% CO2 in air until the two-cell stage by day 1 or blastocyst stage by day 3.5. Thereafter, 20 two-cell embryos were transferred into the oviduct of pseudo-pregnant ICR females at 0.5 dpc. The blastocysts were used for deriving ES cells.
Fibroblasts, bone marrow cells, or ES cells were used for karyotyping. Fibroblasts were derived from mouse tails, which were cut into small pieces and cultured for 7 days. Then fibroblasts or mouse ES cells were incubated with 200 ng/ml demecolcine (Sigma) for 1 h. For bone marrow cells, mice were injected with 15–20 μg demecolcine per mouse and bone marrow cells were isolated 4 h later. The fibroblasts, bone marrow cells, or ES cells were re-suspended in 0.075 M KCl at 37 °C for 10–30 min, followed by carnoy’s fixative (25% acetic acid in methanol) for 30 min cell plating on pre-cleaned slides. For chromosome number counting, the slides were stained with Hoechst 33342. For G banding, the slides were incubated with 0.025% pepsin and then stained with Giemsa for 15 min. More than ten metaphase spreads were analyzed.
Fibroblasts, mouse ES cells, bone marrow cells, or HT-29 cells were harvested, incubated in 0.075 M KCl, and then fixed in 3:1 methanol:glacial acetic acid (v/v) at 4 °C, and dropped onto microscope slides. For embryos, the zona pellucida was removed with Tyrode’s acid and collected onto slides after fixation. The slides were aged at 37 °C overnight, dehydrated through an ethanol series (70, 90, and 100% ethanol for 5 min each) at room temperature and air-dried, and then denatured in 70% formamide/2× SSC at 75 °C for 5 min followed by immediate hydration in a −20 °C precooled ethanol series (100, 90, and 70%). The probe (Additional file 4: Table S9, listed below) was denatured in a water bath at 75 °C for 5 min. The slides were hybridized in a humidified chamber overnight at 37 °C and rinsed 2 × 5 min in 50% formamide/2× SSC at 42 °C, 2 × 5 min in 2 × SSC at 42 °C the following day. Finally, the slides were stained with 10 μL DAPI-antifade solution and mounted with a coverslip. The samples were captured using an Olympus BX53 fluorescent microscope or Nikon Nie-A1 plus fluorescent microscope. To count probe spots in metaphase spreads, an image of DAPI and a merged image of DAPI and probe signal were analyzed together. For counting probe spots in interphase spreads, spots were counted by two individuals.
Derivation of ES cells
Morulae or blastocysts were selected to generate ES cell lines. The zona pellucida was removed using acid Tyrode solution. Each embryo was transferred into one well of a 96-well plate seeded with embryonic fibroblast feeders in ES cell medium supplemented with 20% knockout serum replacement, 1500 U/ml leukemia inhibitory factor (LIF), 3 μM CHIR99021, and 1 μM PD0325901. After 4–5 days in culture, the colonies were trypsinized and transferred to a 96-well plate with a feeder layer in the fresh medium. Clonal expansion of the ES cells proceeded from 48-well plates to six-well plates with feeder cells and then to six-well plates for routine culture.
Derivation of aneuploid ES cells
Mitotic donor cells were obtained after culturing cells with colcemid (75 ng/mL) 10–12 h at 37 °C. Cells were sedimented at 1000 rpm for 10 min and resuspended in 10 ml of chromosome isolation GH buffer. Cells were incubated at 37 °C in a water bath for 10 min and then on ice for 5 min. We added 100 μl of 10% Triton X-100 to cells (final concentration is 0.1%) and incubated them on ice for 5 min. Cells were then lysed by passing three times through a 23G needle. The homogenate was centrifuged at 1000 rpm for 10 min. Supernatant was collected into a new tube and chromosomes were spun down at 2500 rpm for 20 min. Chromosomes were resuspended in 1 ml of HCZB. Zygotes were obtained from the oviducts of superovulated female mice after mating. The chromosomes were microinjected into denuded zygotes using a piezo-driven micropipette 3–4 μm in diameter. Injected zygotes were cultured in vitro until 3.5 dpc in KSOM (aa). Details of the derivation of mouse ES cells are described in the “Derivation of ES cells” section.
Cell culture and transfection
129/Sv × C57BL/6 ES cells were cultured on feeder cells using standard ES cell culture conditions. Cells were transfected with px330 expressing Cas9, mCherry, and sgRNA using Lipofectamine 3000 Reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after transfection, mCherry-positive ES cells were sorted into 96 wells using BD FACS AriaII for further culturing. After 7 days of culturing, the colonies were picked up and expanded for further analysis.
For cell treatment with drugs, the ATM inhibitor KU-55933 (number S1092, Selleckchem) was used at 20 μM. Cells were transfected with plasmids (pX330-mCherry-Ssty2-A and B) and mCherry-positive mouse ES cells were sorted by FACS 12 h after transfection. DNA-FISH analysis was performed 24 or 48 h later.
Human iPSCs were purchased from ATCC (ATCC® ACS-1003™) and cultured on irradiated mouse embryonic fibroblast (iMEFs) feeder layers in serum-free N2B27-LCDM medium as described previously . For transfection, cells were dissociated using TrypLE, replated in iMEF-coated 12-well plates, and transfected in suspension with gRNAs, Cas9, and EGFP or mCherry plasmid using Lipofectamine 3000. Twenty-four hours after transfection, EGFP+/mCherry+ cells were sorted and used for DNA-FISH analysis at 7 days post-transfection.
WGS and off-target analysis
We firstly screened the whole mouse (mm10) and human (hg19) genome for chromosome-specific sgRNAs with our in-house script (https://github.com/pingjie/findChrCrispr). The sgRNAs for each chromosome given by our software are listed in Additional file 1: Tables S2 and S3.
WGS was carried out using Illumina HiSeq X Ten at mean coverages of 20×. Qualified reads were mapped to the mouse reference genome (mm10) by speedseq  (https://github.com/hall-lab/speedseq) with default parameters. FreeBayes (v0.9.10)  and LUMPY  (https://github.com/arq5x/lumpy-sv) were run on the aligned sequence files (BAM files) for short indel detection and structural variation discovery. We firstly filtered germ-line variants which were the same as variants in the “control” (wild-type) samples (untreated mice-XY, untreated mice-XX, untreated mESCs, and TcH14). These results were then filtered to remove variation which overlapped any UCSC repeat regions and microsatellite sequences. The original bam files (pileup) around each candidate variation site were further examined to eliminate those cases where potentially shared variation with “control” samples were not detected by the variant calling software. Next, the raw variant output was manually inspected to remove variants which overlapped with any of the four wild-type samples. For the short indel variations, homopolymers with unit length greater than 2 bp were also removed . Variations after each filtering step are listed in Additional file 1: Table S5.
To search for rearrangements involving on-target DNA sequences that might have integrated elsewhere in the genome, we detected whole genome translocation cases [24, 25] in each sample. After filtering translocations in both mutant and wild-type samples that overlapped with the UCSC repeat regions and microsatellites, the translocations involving the on-target sequences were not observed in any of our mutant samples.
Potential off-target sites of sgRNAs were predicted as previously reported  (http://www.rgenome.net/cas-offinder/). We extracted all the off-target sites with no more than five mismatches for each sgRNA. We searched the short indel variations within the 23-bp predicted off-target sites, and structural variations within 250 bp up- or downstream of the potential off-target sites (Additional file 3: Table S5).
Mouse ES cells were transfected with plasmids carrying indicated CRISPR/Cas9 and were harvested 48 h later and then digested with Protease K to extract for genomic DNA. The iHTGTS libraries were prepared following the protocol described previously with minor modifications . Briefly, linear amplification was performed with 20 nM biotinylated primer (biotin-CCCATTTGCTATTGTTGACAGGAAACCCACTGCC, by Sangon, Shanghai) for 80 cycles; extra primers were removed by 1.2× AxyPrep Mag PCR Clean-Up beads (Axygen, US). Locus-specific primer CTTTGGAGTGAATGTCTGCTCC was used for nested PCR. KpnI was used to block germline fragments, and 1.0× AxyPrep Mag PCR Clean-Up beads (Axygen, US) were used to recover the DNA after enzyme blocking. All the iHTGTS libraries were sequenced by Hiseq. Bioinformatic analysis for off-targets followed the protocol described previously .
Genotyping of the mice was performed by PCR using DNA extracted from their tails. Single ES cell clones were genotyped by nested PCR. The single clone was dissolved in DNA lysis buffer (4 μg/μl proteinase K, 0.1% Triton X 100, and 0.1% Tween 20 in nuclease-free water). The samples were digested at 55 °C for 30 min and then the proteinase K was inactivated at 95 °C for 5 min. PCR was performed using specific primers (Additional file 1: Table S8) under the following conditions: 95 °C for 5 min followed by 35 cycles of PCR (95 °C for 30 s, 58 °C for 30s, and 72 °C for 120 s) for mouse. The nested PCR was 95 °C for 5 min followed by 25 cycles of PCR (95 °C for 30 s, 62 °C for 30s, and 72 °C for 90s) for mouse ES cells. Secondary PCR was performed using 0.5 μg primary PCR product and nested inner primer. PCR was carried out with the same reaction mixture and cycle parameters.
Plasmid DNA delivery into mouse embryos by in utero electroporation
In utero electroporation (IUE) experiments were performed using ICR mice. IUE was performed as previously described . Briefly, E14.5 pregnant ICR mice were anesthetized with sodium pentobarbitone (50 mg/kg), and the uterine horns were exposed. Plasmid mixture (1 μL; containing the px330-EF1α-EGFP-Ssty2-A sgRNA (1 μg/μl), px330-EF1α-EGFP-Ssty2-B sgRNA (1 μg/μl)) with 0.01% Fast Green dye (Sigma)) was injected into the embryos’ lateral ventricle with a glass micropipette. For electroporation, five pulses with a 50 ms duration separated by 950 ms were applied at 32 V using ECM 830 (BTX). Then the uterine horns were placed back into the abdominal cavity to allow the embryos to continue normal development. Forty-eight hours after IUE, the embryos were collected and dissected for FACS.
In summary, we demonstrated that a single specific chromosome, including a sex chromosome and an autosome, could be selectively eliminated via CRISPR-mediated multiple DNA cleavages on the targeted chromosome in culture cells, embryos and in vivo tissues. With the increase of efficiency and specificity, we believe CRISPR/Cas9-Mediated targeted chromosome elimination would be broadly applicable in developing animal models and therapeutic treatments for aneuploidy.
We thank Mu-ming Poo and Dangsheng Li for comments on the manuscript.
This work was supported by National Science and Technology Major Project (2017YFC1001302), CAS Strategic Priority Research Program (XDB02050007, XDA01010409), the MoST863 Program (2015AA020307), NSFC grants (31522037, 31771485), China Youth Thousand Talents Program, Break through project of Chinese Academy of Sciences.
Availability of data and materials
All of the mapped data are available from the SRA under accession SRP070593.
E-WZ designed and performed experiments. X-NH and L-YS performed genotyping, G-band analysis and DNA-FISH. XY and XW derived mouse ES cells and performed ES cell targeting. B-BH, WW, and W-JL performed karyotyping analysis. W-QY transferred embryos. JP and Y.-DS performed data analysis. X-DH, WW, YW and CT constructed plasmids. JY and JH employed iHTGTS to analyzed the genome-wide off-targets. HY supervised the project and designed experiments and HY and M-m. Poo wrote the paper. All authors read and approved the final manuscript.
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).
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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- Driscoll DA, Gross S. Prenatal screening for aneuploidy. N Engl J Med. 2009;360:2556–62.View ArticlePubMedGoogle Scholar
- Matsumura H, Tada M, Otsuji T, Yasuchika K, Nakatsuji N, Surani A, Tada T. Targeted chromosome elimination from ES-somatic hybrid cells. Nat Methods. 2007;4:23–5.View ArticlePubMedGoogle Scholar
- Li LB, Chang KH, Wang PR, Hirata RK, Papayannopoulou T, Russell DW. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell. 2012;11:615–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM, Carone DM, Carone BR, Shivak DA, Guschin DY, et al. Translating dosage compensation to trisomy 21. Nature. 2013;500:296–300.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153:910–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154:1370–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Blasco RB, Karaca E, Ambrogio C, Cheong TC, Karayol E, Minero VG, Voena C, Chiarle R. Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep. 2014;9:1219–27.View ArticlePubMedGoogle Scholar
- Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han YC, Ogrodowski P, Crippa A, Rekhtman N, de Stanchina E, et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature. 2014;516:423–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 2013;41:e141.View ArticlePubMedPubMed CentralGoogle Scholar
- Mahadevaiah SK, Odorisio T, Elliott DJ, Rattigan A, Szot M, Laval SH, Washburn LL, McCarrey JR, Cattanach BM, Lovell-Badge R, Burgoyne PS. Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities. Hum Mol Genet. 1998;7:715–27.View ArticlePubMedGoogle Scholar
- Royo H, Polikiewicz G, Mahadevaiah SK, Prosser H, Mitchell M, Bradley A, de Rooij DG, Burgoyne PS, Turner JM. Evidence that meiotic sex chromosome inactivation is essential for male fertility. Curr Biol. 2010;20:2117–23.View ArticlePubMedGoogle Scholar
- Burgoyne PS, Evans EP, Holland K. XO monosomy is associated with reduced birthweight and lowered weight gain in the mouse. J Reprod Fertil. 1983;68:381–5.View ArticlePubMedGoogle Scholar
- Hunt PA. Survival of XO mouse fetuses: effect of parental origin of the X chromosome or uterine environment? Development. 1991;111:1137–41.PubMedGoogle Scholar
- Probst FJ, Cooper ML, Cheung SW, Justice MJ. Genotype, phenotype, and karyotype correlation in the XO mouse model of Turner syndrome. J Hered. 2008;99:512–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Bondy CA. Turner syndrome 2008. Horm Res. 2009;71 Suppl 1:52–6.PubMedGoogle Scholar
- Donaldson MD, Gault EJ, Tan KW, Dunger DB. Optimising management in Turner syndrome: from infancy to adult transfer. Arch Dis Child. 2006;91:513–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhong Q, Layman LC. Genetic considerations in the patient with Turner syndrome--45, X with or without mosaicism. Fertil Steril. 2012;98:775–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Sheppard O, Wiseman FK, Ruparelia A, Tybulewicz VL, Fisher EM. Mouse models of aneuploidy. TheScientificWorldJOURNAL. 2012;2012:214078.View ArticlePubMedPubMed CentralGoogle Scholar
- Sachidanandam R, Weissman D, Schmidt SC, Kakol JM, Stein LD, Marth G, Sherry S, Mullikin JC, Mortimore BJ, Willey DL, et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature. 2001;409:928–33.View ArticlePubMedGoogle Scholar
- Tomizuka K, Shinohara T, Yoshida H, Uejima H, Ohguma A, Tanaka S, Sato K, Oshimura M, Ishida I. Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci U S A. 2000;97:722–7.View ArticlePubMedPubMed CentralGoogle Scholar
- O’Doherty A, Ruf S, Mulligan C, Hildreth V, Errington ML, Cooke S, Sesay A, Modino S, Vanes L, Hernandez D, et al. An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes. Science. 2005;309:2033–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Iyer V, Shen B, Zhang W, Hodgkins A, Keane T, Huang X, Skarnes WC. Off-target mutations are rare in Cas9-modified mice. Nat Methods. 2015;12:479.View ArticlePubMedGoogle Scholar
- Yang L, Grishin D, Wang G, Aach J, Zhang CZ, Chari R, Homsy J, Cai X, Zhao Y, Fan JB, et al. Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells. Nat Commun. 2014;5:5507.View ArticlePubMedPubMed CentralGoogle Scholar
- Layer RM, Chiang C, Quinlan AR, Hall IM. LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 2014;15:R84.View ArticlePubMedPubMed CentralGoogle Scholar
- Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA, Talkowski ME, Musunuru K. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell. 2014;15:27–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Hu J, Meyers RM, Dong J, Panchakshari RA, Alt FW, Frock RL. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.View ArticlePubMedPubMed CentralGoogle Scholar
- Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol. 2013;31:684–6.View ArticlePubMedGoogle Scholar
- Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, Nezi L, Protopopov A, Chowdhury D, Pellman D. DNA breaks and chromosome pulverization from errors in mitosis. Nature. 2012;482:53–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Adikusuma F, Williams N, Grutzner F, Hughes J, Thomas P. Targeted deletion of an entire chromosome using CRISPR/Cas9. Mol Ther. 2017;25(8):1736–8.View ArticlePubMedGoogle Scholar
- Yang L, Guell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350:1101–4.View ArticlePubMedGoogle Scholar
- Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S, Shelton JM, Bassel-Duby R, Olson EN. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351:400–3.View ArticlePubMedGoogle Scholar
- Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345:1184–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351:403–7.View ArticlePubMedGoogle Scholar
- Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351:407–11.View ArticlePubMedGoogle Scholar
- Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13:659–62.View ArticlePubMedGoogle Scholar
- Pellman D. Cell biology: aneuploidy and cancer. Nature. 2007;446:38–9.View ArticlePubMedGoogle Scholar
- Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Housman DE, Amon A. Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells. Science. 2008;322:703–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ, Brito IL, Hiraoka Y, Niwa O, Amon A. Aneuploidy drives genomic instability in yeast. Science. 2011;333:1026–30.View ArticlePubMedPubMed CentralGoogle Scholar
- Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective antiproliferation compounds. Cell. 2011;144:499–512.View ArticlePubMedPubMed CentralGoogle Scholar
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Heidenreich M, Zhang F. Applications of CRISPR-Cas systems in neuroscience. Nat Rev Neurosci. 2016;17:36–44.View ArticlePubMedGoogle Scholar
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang Y, Liu B, Xu J, Wang J, Wu J, Shi C, Xu Y, Dong J, Wang C, Lai W, et al. Derivation of pluripotent stem cells with in vivo embryonic and extraembryonic potency. Cell. 2017;169:243–57. e225.View ArticlePubMedGoogle Scholar
- Chiang C, Layer RM, Faust GG, Lindberg MR, Rose DB, Garrison EP, Marth GT, Quinlan AR, Hall IM. SpeedSeq: ultra-fast personal genome analysis and interpretation. Nat Methods. 2015;12:966–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv 2016:1207.3907Google Scholar
- Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30:1473–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33:179–86.View ArticlePubMedGoogle Scholar
- Saito T, Nakatsuji N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol. 2001;240:237–46.View ArticlePubMedGoogle Scholar