Generation of sex-reversed female clonal mice via CRISPR/Cas9-mediated Y chromosome deletion in male embryonic stem cells

Abstract

Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR/Cas9-mediated deletion of the Y chromosome allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.

1. Introduction

Genetically modified (GM) animals are essential tools for the study of both fundamental biology and human diseases. The production of GM animals relies on two critical features: (i) stable genome modifications, and (ii) germline transmission of the mutations into a model system. A typical approach for creation of complex GM mice involves the generation of tetra-parental chimeras from normal embryos and GM embryonic stem (ES) cells, followed by multiple rounds of breeding to obtain both male and female homozygotes for germline propagation of the mutations. This process is time-consuming, laborious and costly, particularly if the final objective requires many independent germline manipulations in the same animal.

Mouse ES cells derived from the inner cell mass of blastocysts have unlimited self-renewal and differentiation capacity if maintained in their ground-state pluripotency (Evans & Kaufman, 1981; Martin, 1981; Ying, Wray, Nichols, et al., 2008). Pure ES cell-derived mice (all-ES mice) can be directly and efficiently generated through tetraploid complementation, in which ground-state ES cells are injected into tetraploid blastocysts such that the host 4n cells can only contribute to the extra-embryonic tissue but not somatic tissues (Eggan, Akutsu, Loring, et al., 2001; George, Gertsenstein, Vintersten, et al., 2007; Wen, Saiz, Rosenwaks, et al., 2014). In this system, designed to produce male animals, fertile female all-ES mice (39 chromosome, XO) are occasionally produced from the male ES cell lines (~2%) through spontaneous Y chromosome loss (Eggan, Rode, Jentsch, et al., 2002). Although the monosomic XO female (39, XO) mice have been proposed for the use of GM mice production to avoid mutant allele segregation during outcrossing (Eggan et al., 2002), the observed low frequency makes it impractical for routine use in transgenic facilities.

Here, we present a novel CRISPR/Cas9-mediated approach for directed elimination of the Y chromosome from mouse ES cells permitting efficient generation of monosomic XO female clonal mice by tetraploid complementation. The obtained monosomic XO female clonal mice are viable, fertile, and produce offspring of both sexes when crossed to male clonal mice from the same ES cells.

2. Materials

2.1. Reagents

• CARD HyperOva (#KYD-010-EX, Cosmo Bio)

• Human chorionic gonadotrophin (hCG, #CG10, Sigma-Aldrich)

• KSOM embryo medium (#MR-101-D, Millipore)

• Mineral oil (#ES-005-C, Millipore)

• Knockout DMEM (#10829–018, ThermoFisher)

• Knockout Serum Replacement (KSR) (#10828, ThermoFisher)

• Penicillin-Streptomycin Solution (#TMS-AB-2C, Millipore)

• L-glutamine (Specialty Media, # TMS-001-C, Millipore)

• Nonessential Amino Acids (#TMS-001-C, Millipore)

• Nucleosides for ES cells (# ES-008-D, Millipore)

• β-mercaptoethanol (# ES-007-E, Millipore)

• PD98059 (Cat# V1191, Promega)

• CHIR99021 (#SML1046, Millipore Sigma)

• Recombinant mouse LIF (#ESG1107, Millipore)

• DPBS, no calcium, no magnesium (#14190144, ThermoFisher)

• 0.025% Trypsin and 0.75mM EDTA (# SM-2004-C, Millipore)

• Cell Culture Freezing Medium (# ES-002-D, Millipore)

• DMEM/F-12 (#11320033, ThermoFisher)

• Neurobasal media (#21103049, ThermoFisher)

• N-2 Supplement (#17502048, ThermoFisher)

• B27 Supplement (#17504044, ThermoFisher)

• AlbuMAX I Lipid-Rich BSA (#11020021, ThermoFisher)

• HiFi Cas9 nuclease V3 (#1081060, Integrated DNA Technologies)

• GFP plasmid (#42028, Addgene)

• Neon Transfection System 10μL Kit (#MPK1096, ThermoFisher)

• KAPA Mouse Genotyping Kit (#KK7302, Roche)

• SYBR Green Master Mix (#A25778, ThermoFisher)

2.2. Equipment

• Neon transfection system (#MPK5000, ThermoFisher)

• Cell fusion instrument (#CF-150, BLS)

3. Methods

3.1. Elimination of the Y chromosome in ES cells

• Two crRNAs targeting the Rbmy1a1 gene (See Note 1) are annealed to a tracrRNA at a 1:1:2M ratio to form dual duplex gRNAs by incubation at 95°C for 5min followed by slowing cooling to room temperatures.

• Duplex gRNAs are then incubated with recombinant Cas9 protein at room temperatures for 20min to form gRNA-Cas9 ribonucleoproteins (RNPs), followed by co-delivery with a GFP plasmid (See Note 2).

• ES cells are collected by trypsinization and washed twice with DPBS, then resuspended in supplied R buffer at 100,000 cells/10μL (See Note 3).

• 10μL of cell suspension is mixed with 0.5μL GFP plasmid DNA (See Note 4) and 0.5μL gRNA-Cas9 RNPs. Then 10μL of the mix is loaded to a Neon 10μL tip for electroporation (See Note 5).

• Treated cells are placed in a gelatin-coated 24-well with pre-warmed ES medium and returned to regular culture conditions.

• 48h post-electroporation, cells are digested by trypsin and single GFP+ cells are manually picked with the aid of a micromanipulator under a fluorescent microscope (See Note 6).

• The GFP+ cells are individually plated in 96-well for clonal expansion over feeder layers (See Note 7).

• Use genomic DNA PCR to determine the state of the Y chromosome for presence or loss of the Uba1y and Ssty1genes respectively located to the short and long arm of the Y chromosome (See Notes 8 and 9).

3.2. Generation of fertile female mice from monosomic XO ESCs

• Adult female ICR mice are superovulated and mated individually to males to collect 2-cell embryos (See Notes 10–12).

• 2-Cell embryos are electrofused to generate tetraploid blastocysts for ES cell injection (See Note 13).

• ES cells with confirmed loss of the Uba1y and Ssty1 genes are trypsinized, resuspended in ES medium and kept on ice for blastocyst injection (See Note 14).

• ES cells are picked up in the end of the injection pipette and 10–15 of them are injected into each blastocyst by a flat tip microinjection pipette (See Note 15).

• Before performing embryo transfer, allow injected blastocysts to recover in KSOM for 1–2h (See Note 16).

• Pups obtained from the Y-deletion subclones are all of monosomic XO (39, XO) genotype (See Note 17).

• Distinguish XX and XO female mice by measuring the abundance of the X chromosome- resident Bcor gene relative to Actb in mouse tail genomic DNA using qPCR(See Note 18).

• The fertility of XO female clonal mouse is investigated by breeding with clonal males from the same parental ES cell lines (See Note 19).

5. Concluding remarks

Previous studies demonstrated that targeted chromosomal generation of multiple DNA double-strand breaks (DSBs) using CRISPR/Cas9 can induce directed chromosomal deletion (Adikusuma, Williams, Grutzner, et al., 2017; Warr, Siggers, Bogani, et al., 2009; Zuo, Huo, Yao, et al., 2017). Thus, to eliminate the mouse Y chromosome, RNA-binding motif gene Rbmy1a1 was targeted which has over 50 copies exclusively clustered on the short arm of the Y chromosome (Mahadevaiah, Odorisio, Elliott, et al., 1998). The purified Cas9 proteins with nucleus localization signals can form functional gRNA-Cas9 ribonucleoprotein complexes (RNPs) in vitro. The use of pre-assembled gRNA-Cas9 RNPs allows for more accurate control of RNP composition and doses and has been shown to effectively reduce off-target effects and cytotoxicity in mammalian cells (Kim, Kim, Cho, et al., 2014; Kleinstiver, Pattanayak, Prew, et al., 2016).

Electroporation is widely used to deliver RNPs due to the simplicity and large capacity (Kim & Eberwine, 2010). GFP reporter plasmid with the Cas9 cocktail can be used for validation of successful electroporation. When ES cells with confirmed loss of the Uba1y and Ssty1 genes were used to perform tetraploid complementation, all-ES mice were obtained. The results suggest that the preceding CRISPR/Cas9 manipulation of the ES cells did not adversely affect their pluripotency. Monosomic XO female pups can develop and mature normally to adulthood without noticeable defects. Additionally, no discernible off-targets were found in the XO ESC clones using PCR-based assay via computational predictions to detect the assumptive sites by PCR and sequencing. These results demonstrate the feasibility of efficient deletion of the Y chromosome from mouse ES cells using CRISPR/Cas9 technology allowing generation of male and female clonal mice from the same ES cell line.

Finally, the production of normal XX and XY offspring from XO clonal mice indicates the feasibility to use XO all-ES mice for germline propagation of the complex genetic mutations in mouse models. This system provides a practical strategy to manipulate sex in mice via ES cells, making it possible to expedite the production of complex multi-transgene GM mouse models which is a frequent necessity in current biomedical research, avoiding the time-consuming chimera development step as well as the complex breeding process (Fig. 1).

FIG. 1.

Proposed approach to expedite the production of complex genetically modified (GM) mouse models. (A) Expedite production of GM mice through the generation of XO female GM mice. (B) Overview of the time needed for the proposed approach versus the conventional chimera approach.

Abbreviations

ES

cells embryonic stem cells

GM mice

genetically modified mice