Elimination of the Y chromosome in vitro and in vivo
We initially examined whether complete elimination of a chromosome could be achieved efficiently by using CRISPR/Cas9-mediated multiple cuts at chromosome-specific sites. First, we examined whether the mouse Y chromosome contains unique repeated sequences that could be used for large-scale chromosomal editing via short-guide RNAs (sgRNAs), and whether such editing could result in Y chromosome deletion. Sequence analysis for all mouse chromosomes, using 23-bp sgRNA target sequences containing an adjacent ‘NGG’ protospacer adjacent motif (PAM), showed that each chromosome indeed has unique and multiple repeated sequences for targeting by a single specific sgRNA (Additional file 1: Table S1 and Additional file 2: Table S2). These repeated sequences appeared either clustered at one region or scattered across the entire chromosome (Fig. 1a).
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 [10]. Alternatively, we targeted the spermiogenesis-specific transcript on Y 2 (Ssty2) [11] 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
Next we examined whether this method could be applied to generate animal models for aneuploidy, such as Turner syndrome [1]. We first injected Cas9 mRNA and two specific sgRNAs that targeted the Rmby1a1, Ssty1, or Ssty2 locus into individual mouse zygotes, and injected zygotes were then cultured to the blastocyst stage (Fig. 2a, b). Gene-edited embryos showed normal development compared to untreated embryos (without injection of Cas9 mixture) or embryos treated with two sgRNAs targeting only a single-copy gene (Kdm5d) on the Y chromosome, with a similar blastocyst rate (Fig. 2c). To detect whether the Y chromosome was indeed eliminated, we performed DNA-FISH analysis on injected embryos at the 4- to 16-cell stage. We focused only on male embryos, which were determined by the presence of only a single fluorescent dot for the X chromosome in each blastomere. A green fluorescent signal for the Y chromosome probe was absent in some blastomeres of injected male embryos, suggesting the Y chromosome had been eliminated (Fig. 2d). The efficiency of Y chromosome elimination varied from 40 to 90% among the blastomeres of male embryos from three sets of experiments targeted at three different targeted gene loci (Fig. 2e). Based on the extent of Y chromosome deletion in all blastomeres, the injected male embryos could be classified into three phenotypes: XY (no Y deletion), pure XO (Y deletion in all blastomeres), and XY/XO (Y deletion in some blastomeres) (Fig. 2f). These results indicate that complete or mosaic Y chromosome elimination in mouse embryos could be achieved by this method.
In parallel to the above studies, we transferred the injected zygotes into recipient female mice and obtained newborn mice at similar birth rates compared to control embryos, indicating no developmental defect was induced by the gene editing (Fig. 3a). Interestingly, most of the newborn mice were female, as judged by the presence of female genitals and nipples, with the percentage of females ranging from 79–90% in experiments targeting three different gene loci (Fig. 3b). As a control, mice generated by Cas9 and sgRNA targeting Kdm5d or dCas9 (nuclease-dead Cas9) and sgRNA targeting Ssty2 exhibited normal sex ratios (Fig. 3b). To test whether some of these female mice with Rbmy1a1, Ssty1, or Ssty2 targeting were derived from male embryos that were transformed into females via Y chromosome elimination, we performed karyotyping of tail tissues or bone marrow of all female mice generated by gene editing and found that 26–60% of these mice indeed had 39 instead of 40 chromosomes (Fig. 3c, d; Additional file 1: Table S3). DNA-FISH analysis further confirmed that the missing chromosome was indeed the Y chromosome (Fig. 3e). Furthermore, FISH analysis also showed that some mice exhibited XO and XY phenotypes in different cells of the same mouse, indicating mosaicism in Y deletion (Fig. 3e, f). Further confirmation of complete Y chromosome elimination in XO mice was provided by PCR genotyping, which showed the absence of ten chromosome-specific genes, located in both the short- and long-arm (Fig. 4a, c). In addition, WGS of XO mice also confirmed complete elimination of the Y chromosome (Fig. 4d). Together, these results showed that the Y chromosome could be efficiently eliminated by CRISPR/Cas9-mediated targeting on clustered repeated gene sequences of Rmby1a1 or scattered repeated gene sequences of Ssty1 or Ssty2.
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 [12,13,14]. 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 [17]. Our approach is an efficient way to generate aneuploidy mouse models with mosaicism, which is not found in previous models [18].
The above results on Y chromosome deletion were obtained by using two sgRNAs that target repeat sequences. To generalize this method, we further explored whether the Y chromosome could also be selectively eliminated in zygotes with multiple sgRNAs, each targeting a chromosome-specific single-copy sequence. For this purpose, we designed 14 sgRNAs targeting the short-arm of the Y chromosome (Fig. 5a) and injected Cas9 mRNA together with a cocktail of these 14 sgRNAs into mouse zygotes. We found that 94% of embryos reached the blastocyst stage and 29% of embryos yielded live births after they were transferred into pseudo-pregnant mice, with 73% (69/94) female (76% showed XX karyotypes, 17% showed pure XO karyotypes, and 7% showed XY/XO karyotypes) (Fig. 5b–e; Additional file 1: Table S3), as confirmed by genotyping and DNA-FISH (Fig. 5f–h; Additional file 1: Table S3). Thus, the Y chromosome could also be selectively eliminated in zygotes by CRISPR/Cas9-mediated multiple-sgRNA targeting at chromosome-specific single-copy sequence sites. This approach offers a potential way to use chromosome-specific single nucleotide polymorphisms for chromosome removal without affecting homologous chromosome [19].
Generation of mouse model with Turner Syndrome by X chromosome elimination
Next, we examined whether the same CRISPR/Cas9-mediated genome editing could be used to eliminate the X chromosome. We injected Cas9 mRNA together with a single sgRNA (X-A to X-E) or triple sgRNAs (X-A + B + C and X-C + D + E) that targeted at X chromosome-specific repeated sequences in non-coding DNA sequences (Fig. 6a). We found that triple sgRNAs editing led to serious embryonic lethality, possibly due to large fragment deletion of X chromosomes, whereas single sgRNA targeting yielded some embryos that reached the blastocyst stage (Fig. 6b). We then transferred 2-cell embryos edited with the single sgRNA (X-B, X-C) to recipient female mice and found that these edited embryos yielded birth rates lower than control embryos, using sgRNA that targets at Tyr, a coat color gene with a single copy on autosome (Fig. 6c), suggesting that this gene editing may have induced developmental defects, possibly involving elimination of the single X chromosome in some male embryos or both X chromosomes in some female embryos. Gene-edited newborn female mice consisted of 42.5% XO mice, 55% XX mice and 2.5% XO/XX mice (Fig. 6d, e and Additional file 1: Table S3). The absence of X chromosome in some female mice was confirmed by DNA-FISH and WGS (Fig. 6f, g). As expected, indels or large deletions indeed occurred at targeted non-coding sequences in the remaining X chromosome in XO mice (Fig. 6h, i), but these deletions may not induce obvious deleterious effect (Additional file 1: Figure S2a). In principle, these indels and large deletions could be avoided by sgRNAs that target at only one of the two homologus chromosomes, based on single nucleotide polymorphism. Thus, selective X chromosome elimination could also be achieved by CRISPR/Cas9-mediated gene editing at chromosome-specific repeated sequences, suggesting that this approach could be used for elimination of chromosomes in general.
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
We next tested whether an extra chromosome in aneuploid cells could be eliminated by CRISPR/Cas9 editing. We focused on an ES cell line with an extra human chromosome 14 (hChr14), which was established by chromosome transfer (Fig. 7a, b; see “Methods”) and known to be stable in cell lines [20]. After FACS analysis, we found that 1.6% of the mouse ES cells with hChr14 (termed TcH14) exhibited hChr14 loss during every passage (Fig. 7c, d). Using sgRNAs (14-A to 14-F) targeted at repeated sequence sites, we were able to achieve complete elimination of hChr14 in up to 15% of cells, as indicated by the absence of mCherry expression (Fig. 7c, d). Next, we performed PCR genotyping and DNA-FISH analysis on mCherry-negative clones. We found five out of eight clones from 14-A + F targeting and four out of six clones from 14-F targeting showed complete deletion of hChr14 (Fig. 7e–g). By contrast, clones 14-A + F #2, #6, and #7 and clones 14-F #3 and #6 showed the existence of genes in the short arm and hChr14 DNA-FISH probe, indicating incomplete deletion of hChr14 (Fig. 7f, g). Further genotype evidence of the hChr14 deletion was confirmed by WGS, as well as the expression profile of genes unique to hChr14 (Fig. 7h, i). RNA-seq analysis revealed that all hChr14-specific genes showed no expression in clones 14-A + F #1 and #8. By contrast, ES cells with hChr14 showed a normal expression profile of genes unique to hChr14 (Fig. 7i). In addition, by injecting these aneuploid cells into oocytes and then injecting Cas9 mRNA and sgRNAs 6 h later, we found that 13% of gene-edited blastocysts showed no mCherry signal, indicating complete deletion of hChr14 (Additional file 1: Figure S3a–c). Moreover, we found this method could also be applied to promote human chromosome 7 (hChr7) loss in human cancer cell line HT-29, which contains four hChr7s in most cells (Additional file 1: Figure S4), and extra human chromosome 21 (hChr21) loss in aneuploid mouse ES cell lines (Fig. 8a–e) derived from mice with Down syndrome (DS; Tc1) [21]. Notably, we observed CRISRP/Cas9-mediated multiple DNA cleavages could also produce chromosome rearrangement in cancer cells (Additional file 1: Figure S4e). Finally, we tested whether hChr21 could be selectively deleted via this approach in human iPSCs with trisomy 21 derived from DS patients (ATCC® ACS-1003™). DS iPS cells were transfected with two sgRNAs (21-A and 21-B, containing 49 and 24 cleavage sites, respectively) targeting hChr21-specific repeated sequence sites (Fig. 8a, b). Transfected cells were sorted and analyzed by DNA-FISH with a centromere (CEN) probe for hChr21. We found that 15.0% of cells showed two hChr21 probe signal dots (Fig. 8f–h). As a control, only 6.9% of cells transfected with sgRNA containing only one cleavage site on hChr21 showed two hChr21 probe signal dots (Fig. 8f–h).
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
We next examined whether CRISPR/Cas9-mediated chromosome elimination induces off-target effects in cells and animals. We first analyzed the off-target effects for each sgRNA in seven to nine female mice obtained by Y chromosome elimination (see Additional file 1: Table S3 for corresponding mice). DNA sequencing of PCR products amplified from these genomic sites showed that mutation rarely occurred at all these loci except Ssty1-A (Additional file 1: Figure S5a). We next analyzed off-target sites with up to five mismatches based on WGS described above, including eight mice with Y or X chromosome deletion, and four cell lines with Y chromosome deletion or hChr14 deletion. Among 2186 to 26,469 potential off-target sites for each sgRNA, we found only two off-target sites in only one XO mouse (Ssty1 #1) after several filtering steps as described in previous studies, including ENSEMBL repeats and microsatellites, variation observed in both mutants and controls (Fig. 9a; Additional file 3: Table S5) [22, 23]. The rest of the mice and cell lines contained no off-target mutations (Fig. 9a; Additional file 3: Table S5). We also examined genomic rearrangements, including deletions, duplications, inversions, and copy number variations, using a strategy described in previous reports [24, 25] and found no rearrangements on the on-target DNA sequence and only two rearrangements in only one XO mouse (Ssty2 #1) in the predicted off-targeted sites (Fig. 9b; Additional file 3: Table S5). Furthermore, we examined over 100 metaphase FISH samples among 16 XO mice. We observed that all cells showed XO karyotypes with 39 chromosomes, and none showed fluorescent signals of the Y chromosome probe, suggesting no obvious ectopic translocation of Y chromosome fragments to other chromosomes (Additional file 3: Figure S6).
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) [26] 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
Finally, we continued to explore the molecular mechanism underlying CRISPR/Cas9-mediated targeted chromosome elimination. We first checked whether multiple DNA cleavages on the targeted chromosome and cell division are necessary for chromosome elimination. We treated mouse ES cells or embryos with Cas9 and sgRNA targeting Kdm5d (only one copy on Y chromosome) or dCas9 and sgRNA targeting Ssty2 and found no Y chromosome elimination (Fig. 10a, b). To monitor the process of Y chromosome elimination, we stained the injected embryos at different stages from the one- to eight-cell stage. We found no Y chromosome elimination in one-cell embryos, harvested at 6 h after sgRNA injection (Fig. 10c). However, Y chromosome elimination was observed at the two-cell stage (65%) and increased further at the four- to eight-cell stages (85%) (Fig. 10c). We also tested whether impairing DNA repair by ATM inhibitor KU-55933 could increase the efficiency of chromosome elimination. Mouse ES cells were transfected with Cas9 and sgRNA targeting Ssty2 and then treated with KU-55933. We found that cells treated with KU-55933 for 48 h could increase Y chromosome elimination efficiency by 2.65-fold (Fig. 10d). These results indicate that multiple DNA cleavages on the targeted chromosome, cell division, and DNA repair efficiency are necessary for chromosome elimination.
We next examined whether multiple CRISPR/Cas9-mediated cleavages on a targeted chromosome produce micronuclei, resulting from pulverization of chromosomes [29]. 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.