WO2017000302A1 - 一种孤雄单倍体胚胎干细胞及其制备与应用 - Google Patents

一种孤雄单倍体胚胎干细胞及其制备与应用 Download PDF

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WO2017000302A1
WO2017000302A1 PCT/CN2015/083165 CN2015083165W WO2017000302A1 WO 2017000302 A1 WO2017000302 A1 WO 2017000302A1 CN 2015083165 W CN2015083165 W CN 2015083165W WO 2017000302 A1 WO2017000302 A1 WO 2017000302A1
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library
embryonic stem
semi
dmr
cloned
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李劲松
吴宇轩
钟翠青
尹奇
谢振飞
白梅竹
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Priority to CN201580024974.6A priority patent/CN108368519B/zh
Priority to PCT/CN2015/083165 priority patent/WO2017000302A1/zh
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Definitions

  • the invention relates to biotechnology, in particular to a solitary male haploid embryonic stem cell and preparation and application thereof.
  • RNA interference (RNAi) targeting mRNA levels has now become the best solution for gene function loss screening across mammalian cells.
  • haploid embryonic stem cells (haESCs) (Elling, U., Taubenschmid, J., Wirnsberger, G., O'Malley, R., Demers, SP, Vanhaelen, Q., Shukalyuk, AI, Schmauss, G., Schramek, D., Schnuetgen, F., et al. (2011). Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9, 563-574; Leeb, M., and Wutz, A. (2011). Derivation of haploid embryonic stem cells from mouse embryos. Nature 479, 131-134) provides an ideal tool for genetic analysis.
  • haESCs mammalian haploid embryonic stem cells
  • AG-haESCs solitary male haploid embryonic stem cells
  • ICAMCI intracytoplasmic AG-haESCs
  • DMR differentially methylated region
  • the maternal-derived H19 gene is adjacent to the parent-derived Igf2, which is regulated by the same DMR.
  • This DMR undergoes DNA methylation in the paternal allele and allows only maternal equivalence as a CTCF-dependent insulator.
  • the gene is expressed.
  • the methylation status of this DMR determines whether H19 (H19 expression, DMR demethylation, insulator activation) and Igf2 (Igf2 expression, DMR methylation, insulator inactivation) are expressed.
  • H19 gene or its DMR did not have any serious phenotype in mice (Leighton, PA, Ingram, RS, Eggenschwiler, J., Efstratiadis, A., and Tilghman, SM (1995) .Disruption of imprinting caused by deletion of the H19gene region in mice. Nature 375, 34-39; Thorvaldsen, JL, Mann, MR, Nwoko, O., Duran, KL, and Bartolomei, MS (2002). Analysis of sequence upstream Of the endogenous H19gene reveals elements both essential and dispensable for imprinting. Molecular and cellular biology 22, 2450-2462).
  • This DKO-AG-haESCs efficiently acquires semi-cloned mice (approximately 20% of SC mouse birth rates) and is capable of stably producing multiple genes by genetic manipulation of solitary male haploid embryonic stem cells in vitro. Modified semi-cloned animals. Even more exciting, this DKO-AG-haESCs only requires efficient transfection of a large number of mutant animals in a single step by transfecting a stably expressed sgRNA library and Cas9 at the cellular level in vitro. These experimental results indicate that DKO-AG-haESCs can be used as an intermediate to achieve gene mutations at the individual level by carrying sgRNA libraries, and thus can be further applied to large-scale screening based on gene mutations at the individual animal level.
  • a first aspect of the invention provides a orphan male haploid embryonic stem cell, the H19DMR and IG-DMR genes of the orphan male haploid embryonic stem cell being knocked out.
  • a second aspect of the present invention provides a method for preparing the orphan male haploid embryonic stem cell, comprising knocking out H19 DMR and IG-DMR of a solitary male haploid embryonic stem cell to obtain the orphan male haploid embryonic stem cell.
  • the third aspect of the present invention provides the use of the orphan male haploid embryonic stem cell, which is used for constructing a genetically modified half-gram Long animal.
  • a fourth aspect of the present invention provides a method for constructing a genetically engineered semi-cloned animal, wherein H19 DMR and IG-DMR double knockout solitary male haploid embryonic stem cells are combined with egg cells to obtain a semi-cloned embryo, and the semi-cloned embryo is obtained. Semi-cloned animals.
  • a genetically modified animal which is obtained by the aforementioned method, or a sexually propagated progeny of a semi-cloned animal constructed by the aforementioned method.
  • a method for constructing a genetically engineered semi-clonal animal library of a lone male haploid embryonic stem cell and an sgRNA lentivirus library of the invention is provided.
  • a seventh aspect of the invention provides a genetically engineered semi-cloned animal library obtained by constructing using the aforementioned method.
  • DKO-AG-haESCs can be effectively used for multi-gene genetic manipulation, and further to obtain SC mice carrying polygene genetic modifications by ICAPCI. Furthermore, in combination with sgRNA libraries, DKO-AG-haESCs can be efficiently used to generate heterozygous and homozygous mutant mice in one step, providing further evidence for gene knock-based screening at individual animal levels.
  • DKO-AG-haESCs has several advantages over the existing methods for obtaining genetically modified animals.
  • the current study reports mice that directly inject Cas9 mRNA and sgRNA into a fertilized egg in one step to obtain a transgenic or endogenous gene mutation.
  • This approach demonstrates the potential of CRISPR-Cas9 technology for the preparation of genetically modified animals.
  • DKO-AG-haESCs-mediated gene editing technology provides a unique system in which genetically modified DKO-AG-haESCs may be well analyzed and screened to ensure that the resulting SC animals are expected to inherit Learning the shape, this can greatly avoid the occurrence of chimerism.
  • the injection of traditional diploid embryonic stem cells into blastocysts to obtain genetically modified mice requires delivery through the germline of chimeric mice.
  • the reproductive system is often very time consuming. More seriously, for multi-gene-modified diploid embryonic stem cells, the traits of progeny are separated during the transmission of the germline, and only a small number of offspring may contain all the desired genetic modifications.
  • DKO-AG-haESCs can achieve F0 mice with multiple gene modifications in one step.
  • all ESC-derived mice can be obtained in one step by injecting diploid embryonic stem cells into tetraploid blastocysts (also known as tetraploid compensation techniques).
  • This method is also widely used as the most stringent standard for testing the pluripotency of reprogrammed cells such as induced pluripotent embryonic stem cells (iPSCs) and nuclear transfer embryonic stem cells (ntESCs).
  • iPSCs induced pluripotent embryonic stem cells
  • ntESCs nuclear transfer embryonic stem cells
  • embryonic stem cells or animals that induce pluripotent embryonic stem cell origin are very low in birth efficiency, probably due to the epigenetic instability of diploid embryonic stem cells.
  • DKO-AG-haESCs can be obtained stably and efficiently after long-term in vitro culture, especially after genetic manipulation.
  • ICAPIC technology is similar to ROSI.
  • ROSI is a mature technology in different mammals, including non-human primates and humans.
  • cynomolgus-derived haploid embryonic stem cell lines have been successfully established, and ICAPIC technology may be used in the near future to obtain genetically modified non-human primates efficiently and rapidly.
  • DKO-AG-haESCs can pass sgRNA The library obtained a large number of genetically altered animals.
  • this method is more simple and labor-saving than direct injection of plasmid or mRNA into the embryo, because the DKO-AG-haESCs carrying the Cas9 and sgRNA libraries can be used repeatedly as injections.
  • Body to continuously and efficiently produce different genetically modified animals.
  • These different mutant animals can be used to identify sgRNA insertions (like a bar code for mutant animals) by a universal primer PCR simply and quickly.
  • a method of injecting a plasmid or mRNA directly into an embryo requires separately preparing each separately injected sgRNA separately. The obtained mutant animals need to be cultured separately, and subsequent identification requires the use of corresponding primers for specific genes.
  • sgRNA library such as some differentially expressed genes obtained by high-throughput analysis at the cellular level
  • a sub-library of sgRNAs may be made, perhaps these genes will participate in a particular stage of the developmental process.
  • important genes associated with this developmental process can be screened at an individual level quickly and efficiently.
  • DKO-AG-haESCs mediate the acquisition of SC animals, which is an effective and simple method to generate genetically modified animal models carrying multiple gene mutations or multiple gene knock-ins in gene families. Furthermore, in combination with sgRNA libraries, DKO-AG-haESCs are capable of obtaining large-scale genetically modified animals in one step. Although the mechanism by which DKO-AG-haESCs can efficiently acquire the presence of SC animals is still curious, this approach may be able to facilitate further exploration of developmental processes and complex diseases.
  • Figure 1A is a schematic representation of SC mice obtained by intracytoplasmic injection of two DMR knockout AG-haESCs (ICAHCI).
  • ICAHCI DMR knockout AG-haESCs
  • Figure 1B Sperm injection into the enucleated MII egg to obtain a lone male haploid embryo.
  • Figure 1C One of the solitary male haploid embryonic stem cells established by the solitary haploid blastocyst.
  • Figure 1D is established after multiple flow enrichment of haploids Lone male haploid embryonic stem cell
  • Figure 1F shows the sgRNA sequence of IG-DMR knocked out
  • Figure 1G obtained Cell line. Left side: mCherry positive cells were collected and plated into culture plates. Right side: established Cell line
  • Figure S1B More than 80% of the cells reconstituted with embryos can reach 2-cells, and the developmental efficiency of spherical sperm injection (ROSI) is similar.
  • FIG. 1C The 3 cell line obtained SC mice by ICAMCI.
  • the asterisk represents a SC mouse with birth retardation.
  • FIG. 1E Cell-derived normal and retarded SC mice were subjected to expression analysis of imprinted genes (Gtl2 and Dlk1) on different organs.
  • Figure 2A The semi-cloned mouse obtained from the cell line (p17), the asterisk represents a growth-blocking mouse, and will die shortly after birth.
  • Figure 2B H19 DMR methylation status of normal SC mice and abnormal SC mice obtained from cell lines.
  • FIG. 2C Schematic diagram of the design of H19 DMR knockout sgRNAs.
  • Figure 2D obtained Cell line genotype identification, these cell lines were obtained by knocking out H19 and IG-DMRs on the AGH-OG3 cell line.
  • Figure 2E Genotyping of progeny produced after mating of SC mice obtained by DKO-AG-haESCs, as long as the offspring carrying IG-DMR will die before or shortly after birth.
  • Figure 2F analyzes the gene expression profile of DKO-AG-haESCs by RNA-seq.
  • the expression profile of a gene is the clustering of the expression of all genes.
  • These three DKO-AG-haESCs cell lines showed similar gene expression profiles to the control group AG-haESCs, but were very different from spherical sperm.
  • FIG. 2G Imprinted gene expression profile of DKO-AG-haESCs.
  • the expression profiles of three different DKO-AG-haESCs were similar to those of the control group, but different from the spherical sperm of mice.
  • Figure 2H analyzes the methylation status of DKO-AG-haESCs by RRBS.
  • Figure S2A Cell knockout obtained by IG-DMR Cell lines were genotyped.
  • FIG. 3A Schematic diagram of Tet1, Tet12 and Tet13sgRNA
  • Figure 3B FACS collects mCherry-positive cells and plated them in culture dishes to obtain DKO-AG-haESCs with Tet family gene mutations.
  • Figure 3C shows the Tet-TKO-DAH cell line.
  • Figure 3F Schematic representation of p53, p63 and p73sgRNA.
  • FIG. 3H Schematic representation of Tet1-EGFP, Tet2-mCherry, Tet1-ECFP double-stranded DNA vectors.
  • EGFP, mCherry, and ECFP were fused to the stop codons of Tet1, Tet12, and Tet13, respectively.
  • Figure S3A Two DKO-AG-haESCs cell lines obtained by knocking out H19 and IG-DMR on WT-AG-haESCs (AGH-OG3) ( with ) Sequencing analysis.
  • FIG. 1 Figure S3D Expression analysis of DKO-AG-haESCs and normal AG-haESCs imprinted genes (H19, Igf2, Gtl2 and Dlk1). After H19 and IG-DMR knockdown, the expression levels of H19 and Gtl2 of AGH-OG-3 were down-regulated. In contrast, the expression levels of Igf2 and Dlk1 were up-regulated.
  • Figure S3F The methylation levels of the H19-Igf2 and Dlk1-Dio3 imprinted cluster regions.
  • Figure 4A is a schematic representation of a large number of heterozygous mutant SC mice obtained by DICA-AG-haESCs carrying a sgRNA library via ICAMCI.
  • Figure 4B represents the enrichment of mc9 successfully transferred into expressed mCherry positive haploids by FACS and used in subsequent IACHCI.
  • Figure 4C shows sgRNA PCR identification of single cell-derived haploid clones. All cell clones tested were sgRNA.
  • Figure 4D sequenced different mutant genes in cell clones. All of the clones tested were modified with the gene of interest.
  • Figure 4E was derived from SC mice carrying the sgRNA library DKO-AG-haESCs.
  • FIG. 4G Mutation sequencing of different genes of interest in SC mice. All of the tested SC mice were modified with the gene of interest.
  • Figure S4E The p53, p63 and p73 genes were sequenced from SC mice obtained from the p53-TKO-DAH-1 and p53-TKO-DAH-2 cell lines, respectively.
  • Figure 5A is a schematic representation of a large number of biallelic mutant SC mice obtained by IKACI carrying DKO-AG-haESCs that continuously express Cas9 and sgRNA libraries.
  • FIG. 5B PCR analysis of single cell derived cell clone Cas9. All cell clones tested contained the Cas9 transgene.
  • Figure 5D shows sgRNA PCR identification of single cell derived cell clones.
  • Figure 5E was derived from SC mice carrying DKO-AG-haESCs that continuously expressed Cas9 and sgRNA libraries.
  • Figure 5F PCR identifies sgRNA of SC mice.
  • Figure 5G carries DKO-AG-haESCs that continuously express Cas9 and sgRNA libraries to obtain biallelic mutant SC mice via ICAMCI. Take the Polm biallelic mutant SC mouse as an example.
  • Figure 5H analyzes the mutation of the Polm gene in the rat tail by TA cloning and sequencing. A frameshift mutation occurred in 24 of the 26 detected clones.
  • Figure 5I Summary of TA clone sequencing results for 7 biallelic mutant SC mice. More than 80% of clones have insertional deletion mutations.
  • Figure 5J Scube1 biallelic mutant SC mice were subjected to TA cloning and sequencing analysis of their different organs.
  • FIG. 5A Schematic representation of Tet1-EGFP, Tet2-mCherry and Tet3-ECFP exogenous double stranded vectors.
  • Figure S6A Transiently transfected pX330-mCherry plasmid and haploid cells carrying sgRNA into mature egg cells, followed by injection of Cas9 mRNA into the reconstituted egg cells (this program name: Lenti-sgRNA + pX330 + Cas9 injection).
  • Figure S6B Single cell-derived haploid ES clone PCR to identify its sgRNA insertion.
  • the present invention first provides a orphan male haploid embryonic stem cell in which H19 DMR and IG-DMR of the orphan male haploid embryonic stem cell are knocked out.
  • the whole genome of the orphan male haploid embryonic stem cells is derived from sperm, and has the self-replication ability of stem cells and more Energy, can replace the sperm and oocyte binding to support the complete development of the embryo.
  • the H19 DMR refers to a section of the M19-Igf2 imprinted cluster (DMR: differentially methylated region).
  • DMR differentially methylated region
  • the specific location and sequence of the H19 DMR can be determined by methods such as existing methylation sequencing or homologous sequence analysis prediction. It is known that the human H19 DMR is located in the chromosome 11p15.5 region, and the mouse H19 DMR is located at the distal end of chromosome 7, between the two genes H19 and Igf2, and the upstream of the H19 gene from the 2kb to 4kb position.
  • H19 DMR is methylated in the paternal allele, resulting in the inability of CTCF protein to bind to this methylation region, so that the enhancer downstream of H19 does not need to overcome the CTCF barrier, thereby enhancing the expression of upstream Igf2.
  • CTCF protein can bind to this unmethylated region, so the enhancer downstream of H19 can only enhance the expression of H19, but can not regulate the upstream Igf2. If the paternal H19DMR is knocked out, then the enhancer downstream of H19 can upregulate the expression of Igf2.
  • the solitary male haploid is a paternal source, it should theoretically be completely methylated, but the study found that the methylation of the lone male haploid H19 DMR in vitro was abnormally erased and demethylated. The state results in abnormal up-regulation of H19 expression and down-regulation of Igf2 expression.
  • the present invention knocks out H19 DMR and corrects the abnormal state in which H19 expression up-regulates Igf2 expression down-regulation.
  • the IG-DMR refers to a section of the Dlk-Dio3 imprinted cluster (DMR: differentially methylated region).
  • DMR Dlk-Dio3 imprinted cluster
  • the specific location and sequence of IG DMR can be determined by methods such as existing methylation sequencing or homologous sequence analysis prediction. It is known that the mouse IG-DMR is located on chromosome 12, which is a 4.15 kb repeat between Dlk1 and Gtl2 genes in the imprinted cluster, and human is located on chromosome 14 (14q32.2). In the paternal allele of IG-DMR, DNA methylation occurs in this region. The gene Gtl2 and some mircroRNAs of this imprinted cluster are not expressed, but the genes Rtl1, Dlk1 and Dio3 are expressed.
  • orphan male haploid embryonic stem cells are subjected to other genetic modification in addition to H19 DMR and IG-DMR knockout.
  • Genetic modification specifically refers to the structural changes in genes compared to before transformation by biological or chemical or physical means. This change primarily refers to changes in base pair composition, including but not limited to changes in one or more base pair substitutions, additions, and deletions.
  • the orphan male haploid embryonic stem cells are derived from a mammal and may be human or non-human mammals.
  • the orphan male haploid embryonic stem cells are derived from rodents, such as rabbits and mice, and the mice may be mice or rats.
  • the orphan male haploid embryonic stem cells are derived from a mouse.
  • the orphan male haploid embryonic stem cells of the present invention have a higher birth rate in the construction of semi-cloned animals.
  • the invention also provides a method for preparing the orphan male haploid embryonic stem cells, comprising knocking out the H19 DMR and IG-DMR of the orphan male haploid embryonic stem cells to obtain the orphan male haploid embryonic stem cells.
  • the H19 DMR and IG-DMR can be knocked out using existing gene editing methods.
  • the H19 DMR and IG-DMR are knocked out using CRISPR/Cas9 mediated genetic manipulation.
  • Other methods can also be used to achieve gene knock The method is not limited to the ones listed in the examples.
  • H19 DMR Based on the knockout of H19 DMR, the complete sequence of H19 DMR was removed from the chromosomal DNA; based on the knockout of IG DMR, the complete sequence of IG-DMR was removed from the chromosomal DNA.
  • the H19 DMR knockout orphan male haploid embryonic stem cells are first constructed and the IG-DMR is further knocked out on the basis of this.
  • the IG-DMR knockout orphan male haploid embryonic stem cells are first constructed and, based on this, the H19 DMR is further knocked out.
  • lone male haploid embryonic stem cells that are simultaneously knocked out by H19 DMR and IG-DMR are directly constructed.
  • orphan male haploid embryonic stem cells are also genetically modified.
  • the other genetic modification described refers to genetic modification other than H19 DMR and IG-DMR double knockout.
  • the other genetic modification can be the engineering of a single target gene or the modification of multiple target genes of interest.
  • Target genes of interest are not specific and can be set and modified according to research needs. For example, it can be a modification of one, two, or more than three target genes. Since the H19 DMR and the IG-DMR double knockout of the solitary male haploid embryonic stem cells of the present invention can be passaged in vitro, it can be genetically modified in theory, and the number of the target genes can be manipulated as needed, without particular limitation. .
  • Such genetic engineering includes, but is not limited to, knock-in of a target gene, knockout of a target gene, and the like.
  • Knock-in of target genes and knockout of target genes can be accomplished by techniques such as gene targeting and homologous recombination, including but not limited to ZFN (zinc finger nuclease), TALEN (transcriptional activation-like effector nuclease), and CRISPR/ Genetic manipulation of Cas9 (clustered regular interval short palindromic repeat technique).
  • the H19 DMR and IG-DMR double knockout solitary male haploid embryonic stem cells are further subjected to single or multiple genetic engineering to obtain H19 DMR and IG-DMR double knockout and other genetically modified orphans.
  • Male haploid embryonic stem cells Alternatively, the orphan male haploid embryonic stem cells can be genetically engineered, and then the H19 DMR and IG-DMR of the mutant maleloid embryonic stem cells are knocked out.
  • any other biotechnological means that can achieve H19 DMR and IG-DMR double knockout and other gene mutations can be used to construct H19 DMR and IG-DMR double knockout and other genetically modified solitary male haploid embryos. stem cell.
  • the invention also provides the use of the orphan male haploid embryonic stem cells for the construction of genetically engineered semi-cloned animals.
  • the orphan male haploid embryonic stem cells are used as a fertilization medium to construct a genetically modified animal.
  • the invention also provides a method for constructing a genetically modified semi-cloned animal, which comprises combining H19 DMR and IG-DMR double knockout solitary male haploid embryonic stem cells with egg cells to obtain a semi-cloned embryo, and cultivating the semi-cloned embryo to obtain a semi-cloning animal.
  • the egg cells and the orphaned haploid embryonic stem cells are derived from the same type of animal, preferably the same species.
  • the semi-cloned embryos can be H19 DMR and IG-DMR double knockout solitary male haploid embryonic stem cells as donors of ICAMCI, and semi-cloned embryos were obtained by ICAPCI method.
  • the semi-cloned embryos can be obtained by culturing the semi-cloned embryos in a suitable mother by a method of embryo transfer.
  • the suitable parent is a pseudopregnant ICR mother.
  • orphan male haploid embryonic stem cells are engineered by other genes.
  • the present invention also provides a genetically modified animal, which is obtained by the aforementioned method, or is a sexually propagated progeny of a semi-cloned animal constructed by the aforementioned method.
  • the semi-cloned animal of the present invention may be a non-human mammal.
  • the semi-cloned animal is a rodent such as Rabbit, mouse.
  • the semi-cloned animal is a mouse.
  • the invention also provides a method of constructing a genetically engineered semi-clone animal library comprising the following steps:
  • the sgRNA lentiviral library plasmid contains several lentiviral vectors expressing different sgRNAs.
  • the sgRNA lentiviral library can be constructed using existing techniques, as well as existing sgRNA lentiviral library plasmids. Specifically, sgRNA designed for different genes can be cloned into a lentiviral vector. The sgRNA can be designed according to the gene of interest.
  • a commercially available mouse whole genome sgRNA lentivirus library is employed.
  • step 2) may be selected from any of the following:
  • the lone male haploid embryonic stem cell library carrying the sgRNA library was further transfected with the plasmid expressing Cas9, and the obtained lone male haploid embryonic stem cells were used as donors of ICAMCI, and the semi-cloned embryos were obtained by ICAPCI method.
  • Lone male haploid embryonic stem cells carrying a solitary male haploid embryonic stem cell library carrying sgRNA library were used as donors of ICAMCI, and mature eggs were injected by ICAPCI method, and Cas9 mRNA was injected into the reconstructed egg to obtain semi-cloned embryos.
  • the sol-bearing haploid embryonic stem cell library carrying the sgRNA library was further transfected into the plasmid expressing Cas9, and the obtained solitary male haploid embryonic stem cells were used as donors of ICAMCI, and the mature eggs were injected by ICAPCI method, and then the reconstructed eggs were injected.
  • Semi-cloned embryos were obtained by injecting Cas9 mRNA, and semi-cloned animals were obtained by embryo transfer.
  • the Cas9-expressing plasmid described in the above methods A and C can be cloned into an expression plasmid construct.
  • the Cas9 expressing plasmid is a pX330-mCherry plasmid. Plasmids that can be used to construct Cas9 expression are not limited to the pX330 plasmid. The expression plasmid only needs to be suitable for expression of a foreign gene in mammalian cells.
  • the invention also provides another method of constructing a genetically engineered semi-cloned animal library comprising the following steps:
  • lentiviral particles prepared by expressing Cas9 lentiviral particles and sgRNA lentiviral library are infected with the lone male haploid embryonic stem cells of the present invention, and a lone male haploid embryonic stem cell library carrying sgRNA library and Cas9 sustained expression is obtained;
  • the Cas9-expressing viral particle can be obtained by cloning a gene encoding Cas9 into a lentiviral vector and then performing lentiviral packaging using a prior art technique, and a lentiviral vector expressing Cas9 is commercially available.
  • the semi-clone animal library contains several genetically modified semi-cloned animals.
  • the animal can be heterozygous or double allele Mutant mutant animal.
  • the semi-cloned embryos can be obtained by culturing the semi-cloned embryos in a suitable mother by a method of embryo transfer.
  • the appropriate parent can be selected from a pseudopregnant ICR mother.
  • the invention also provides a genetically engineered semi-cloned animal library obtained by constructing using the aforementioned method.
  • the genetically engineered semi-clonal animal library of the present invention can perform genetic screening of genes at the level of subordinate individuals.
  • the semi-clone animal library of the present invention may be a non-human mammalian library.
  • the library of semi-cloned animals is a rodent library, such as a rabbit library, a murine library.
  • the library of semi-cloned animals is a mouse library.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature.
  • AG-haESCs Lone male haploid embryonic stem cells
  • DKO-AG-haESCs H19 DMR and IG-DMR double knockout of solitary male haploid embryonic stem cells
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • KSR serum replacement
  • trypsin Opti-MEM
  • DPBS DPBS
  • Lipofectamine 2000 were purchased from Life Technologies; restriction enzymes, T4 ligase were purchased from NEB. The company; Taq enzyme and dNTPs were purchased from TaKaRa; CDNA reverse transcription kit, fluorescent quantitative reagent SYBR-Green was purchased from TOYOBO; and oligonucleotides were synthesized by Shanghai Generay.
  • Activation fluid 10 mM Sr2+, 5 ng/ml Trichostatin A (TSA)
  • DMEM millipore, CAT#SLM-220-M 75%, 20% serum replacement KSR (Gibco, CAT#10828-028), 1,500 U/ml LIF (Millopre, CAT#ESG1107), 3M CHIR99021 (Stemgent, CAT #04-0004) and 1M PD0325901 (Stemgent, CAT#04-0006)
  • the px330 (addgene) plasmid was digested with NotI, and then the CMV-mcherry-pA fragment amplified from pmCherry-C1 (Clontech) was inserted into the digested px330 plasmid.
  • mCherry-F ATTTGCGGCCGCATAGTAATCAATTACGGG
  • mCherry-R ATTTGCGGCCGCATGCAGTGAAAAAAATGC
  • the mouse testis was digested with collagenase IV for 20 min, digested with trypsin for 10 min, and then spherical sperm of the mouse was obtained by FACS (flow sorting).
  • H19 ⁇ 3.8 kb KO mouse C57/B6 background, homozygous: Reference construction (Thorvaldsen, JL, Mann, MR, Nwoko, O., Duran, KL, and Bartolomei, MS (2002). Analysis of sequence upstream Of the endogenous H19 gene reveals elements both essential and dispensable for imprinting. Molecular and cellular biology 22,2450-2462.
  • IG-DMR KO mice C57/B6 background, heterozygous: Reference construction (Lin, SP, Youngson, N., Takada, S., Seitz, H., Reik, W., Paulsen, M., Cavaille , J., and Ferguson-Smith, AC (2003). Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12.Nature genetics 35,97-102,)
  • B6D2F1 (C57BL/6X DBA2) female mice: Female offspring obtained after mating with male mice of the C57BL/6 strain and males of the DBA2 strain.
  • ICR adult rats were purchased from Slack, and ICR adult female rats were mated with ligation ICR male mice to obtain ICR pseudo-pregnant female rats.
  • Mouse MII eggs were harvested 14 hours after human chorionic gonadotropin (HCG) treatment and then enucleated in a HEPES-CZB medium containing 5 ug/ml cytochalasin B (CB) using a Piezo needle. After enucleation, a single sperm head is injected into the cytoplasm of the egg.
  • the reconstituted embryos were cultured in CZB medium for 1 hour and then transferred to an activation solution containing 1 mM Sr 2+ for activation. After activation, all reconstituted embryos were transferred to KSOM medium containing amino acids and cultured at 37 ° C, 5% CO 2 . Reconstituted embryos that reached the mulberry or blastocyst stage after 3.5 days were planted in ESC medium.
  • the reconstructed embryonic zona pellucida was digested and removed by Acid Tyrode solution. Each was transferred to a 96-well plate well plated with mouse fibroblast trophoblast and cultured with ESC medium containing 20% serum replacement (KSR), 1,500 U/ml LIF, 3M CHIR99021 and 1M PD0325901. After 4-5 days of culture, the cell clones were trypsinized and passed to 96-well plates plated with fresh trophoblasts. The cells were further expanded and passaged into 48-well plates and further into 6-well plates, and the daily cells were maintained in only 6-well plates.
  • KSR serum replacement
  • haploid cells To sort out haploid cells, the embryonic stem cells were trypsinized and washed once with PBS (GIBCO) and then in ESC medium containing 15 ⁇ g/ml Hoechst 33342. Water bath for 30 min. Subsequently, cells of haploid 1N peak shape were collected by flow sorter BD FACS AriaII for subsequent culture to obtain a lone male haploid embryonic stem cell line.
  • CRISPR-Cas9 plasmid construction annealing the forward oligonucleotide strand and the reverse oligonucleotide strand of the synthesized sgRNA to obtain a double-stranded oligonucleotide strand (the sgRNA sequence in the present invention refers to the positive oligo of the sgRNA)
  • the nucleotide sequence was then ligated to pX330-mCherry, which was digested with BbsI (New England Biolabs).
  • BbsI New England Biolabs
  • the corresponding plasmids constructed were transfected into solitary male haploid embryonic stem cells using Lipofectamine 2000 (Life Technologies) according to the instructions.
  • haploid cells with red fluorescent protein were sorted by flow (FACS Aria II, BD Biosciences) and plated at a lower density. After 4-5 days of growth, the monoclonals were picked for subsequent construction. Finally, the cell line of the corresponding gene mutation is obtained by sequencing the PCR target gene.
  • Double-stranded DNA donor preparation
  • the sequence encoding EGFP, mCherry or ECFP was amplified and ligated to the pMD19-T vector accordingly to obtain pMD19-T-EGFP/mCherry/ECFP. Subsequently, the left and right homology arms of the gene of interest were inserted correspondingly into the vector of pMD19-T-EGFP/mCherry/ECFP.
  • Mouse sgRNA virus library and Cas9 virus plasmid have been reported (Cong, L., Ran, FA, Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, PD, Wu, X., Jiang, W., Marraffini, LA, et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823; Koike-Yusa, H., Li, Y., Tan, EP, Velasco -Herrera Mdel, C., and Yusa, K. (2014). Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.
  • mouse sgRNA virus used in the present invention The library and the viral plasmid of Cas9 were supplied by Addgene.
  • HEK293T was passaged into a 10 cm culture dish in advance. 2000 Reagent (Invitrogen, Life Technologies) was transfected into HEK293T cells transfected with 3 ug of viral plasmid (sgRNA lentivirus library or Cas9 lentivirus) and 9 ⁇ g of ViraPower Lentiviral Packaging Mix lentiviral packaging mix (Invitrogen). After 72 hours of transfection, the supernatant was collected and concentrated with Lenti-Concentin virus precipitation solution (SBI), and then stored at -80 °C.
  • SBI Lenti-Concentin virus precipitation solution
  • Cas9 Lentivirus infection A cell suspension containing 108 DKO-AG-haESCs was infected with 8 ug/ml polybrene (Sigma) and packaged Cas9 lentivirus for 48 hours, then dosed with 10 ug/ml blasticidin (Sigma) After 3 days of screening, the remaining drug resistant clones were Cas9 lentivirus-integrated cell lines.
  • sgRNA Lentiviral Library Infection Cell suspension containing 108 DKO-AG-haESCs was infected with 8 ug/ml polybrene (Sigma) and packaged CRISPR-sgRNA library lentivirus, and changed to 1 ug/ml puromycin (Invitrogen) 48 hours later The medium was subjected to drug screening, and the positive result obtained after 2 days of screening was a cell line carrying lentiviral integration of the sgRNA library.
  • the Cas9 lentivirus-integrated cell line was prepared first, and then the sgRNA lentivirus library was further infected, and the drug was screened in a medium of 1 ug/ml puromycin (Invitrogen). The positive result after screening for 2 days was Cas9 lentivirus and sgRNA library lentivirus. Integrated cell line.
  • the DKO-AG-haESCs cell line is only infected with the sgRNA lentivirus library, then the cells do not have Cas9 expression. At this time we need to transfect the pX330-mCherry plasmid at the cellular level to achieve genome editing. 1.6 sulfite methylation sequencing
  • mice DNA was coated with 15 ul of 2% LMP agarose (low melting point agarose) into beads, 460 ul of DNA digestion buffer was added, and 40 ul of proteinase K was added to each sample, and the sample was incubated at 50 ° C overnight.
  • LMP agarose low melting point agarose
  • EZ DNA methylation Gold kit ZYMO Research
  • the obtained kit recovers the product, and uses this as a template for PCR, product recovery, connection with PMD19-T carrier, transformation, and plating.
  • the 10 bacterial cells of the plate were selected for sequencing.
  • RNA of cells or organs was extracted with Trizol reagent (Invitrogen), and then reverse transcribed into cDNA using 1 ug of total RNA using a First Strand cDNA Synthesis kit (TOYOBO).
  • Real-time PCR reactions were performed using SYBR Green Realtime PCR Master Mix (TOYOBO) and performed on a Bio-Rad CFX96 instrument with 3 replicates per set of samples. All gene expression levels were based on the expression level of the housekeeping gene Gapdh.
  • Intracytoplasmic AG-haESCs injection ICAHCI
  • AG-haESCs were treated with medium containing 0.05 ⁇ g/ml colchicine for 8 h to synchronize cells to M phase, followed by cytoplasmic injection.
  • the digested AG-haESCs were washed 3 times with HEPES-CZB culture medium, and then resuspended in 3% (w/v) polyvinylpyrrolidone (PVP) HEPES-CZB medium.
  • PVP polyvinylpyrrolidone
  • the nucleus of each M phase of AG-haESCs was injected into the MII egg using a Piezo micromanipulator.
  • the reconstructed embryos were first incubated in CZB culture for 1 h and then activated with CB-free activation for 5-6 h. After activation, all reconstituted embryos were cultured in KSOM medium at 37 ° C, 5% CO 2 .
  • ICAHCI embryos can be cultured in KSOM for 24 h to reach 2-cell stage embryos
  • ROSI spherical sperm injection
  • Each 15-20 2 cell embryos obtained from ICAMCI or ROSI were transferred to each uterus of 0.5 dpc (0.5 days after mating) pseudopregnant ICR mice.
  • Mother rats undergo caesarean section or natural production after 19.5 days of pregnancy.
  • Caesarean section is a reconstructed embryo obtained from WT AG-haESCs or a single DMR knockout of AG-haESCs, and the expired fetus is rapidly stripped from the uterus of the mother.
  • the mothers who are pregnant for 19.5 days can be naturally produced. After removing the fluid from the born mice, the mice are placed in an oxygen-containing incubator, and the surviving mice are subsequently raised by the surrogate mother.
  • RNA-seq library of total RNA was based on Illumina's official TreSeq RNA Sample Prep v2Guide. Upon completion, deep sequencing was performed on the Illumina HiSeq 2000 instrument of the Computationalomics Center of the Institute of Computational Biology, Chinese Academy of Sciences. 2 WT AG-haESCs, And a total of 6 samples of spherical sperm for subsequent analysis
  • the gene expression amount is RPKM, and the specific algorithm is (Yang, L., Duff, MO, Graveley, BR, Carmichael, GG, and Chen, LL (2011). Genomewide characterization of non-polyadenylated RNAs. Genome biology 12, R16) is consistent.
  • the p-value of the differentially expressed gene was used to calculate the waldscore method (Yang et al., Genome boil 2011), and abs (waldscore) > 1.96 (i.e., p-value ⁇ 0.05) was selected, and then the differentially expressed genes were selected.
  • the construction of the RRBS is based on the official standard of Illumina. Then sequencing with Illumina HiSeq 2000 (Gu, H., Smith, ZD, Bock, C., Boyle, P., Gnirke, A., and Meissner, A. (2011). Preparation of reduced representation bisulfite sequencing libraries for genome -scale DNA methylation profiling.Nature protocols 6,468-481.). All sequencing reads were compared to the mouse genome for analysis.
  • the extracted genomic DNA was subjected to PCR using the corresponding primers, and then the PCR product was further subjected to agarose gel electrophoresis analysis.
  • the haploid sperm head of H19 ⁇ 3.8 kb mice was injected into the enucleated egg cells (Fig. 1A) by the method of the above 1.3 to obtain reconstructed blastocysts, and a lone male haploid embryonic stem cell line was constructed.
  • Three haploid cell lines were established from 250 reconstituted blastocysts (named ) (Fig. 1B-1D).
  • mice were constructed using wild-type AG-haESCs as donors to ICAMCI. Normal half-clone mice have a birth efficiency of approximately 0.7-1.8%.
  • Gtl2-F TTGCACATTTCCTGTGGGAC
  • Gtl2-R AAGACACCATAGGCCACTAGG
  • IG-DMR-sgRNA1 and IG-DMR-sgRNA2 Two sgRNAs (designated IG-DMR-sgRNA1 and IG-DMR-sgRNA2) knocked out of 4.15 kb IG-DMR were designed between Dlk1 and Gtl2 (Fig. 1F).
  • IG-DMR-sgRNA1 sequence CGTACAGAGCTCCATGGCAC (SEQ ID NO: 1)
  • IG-DMR-sgRNA2 sequence CTGCTTAGAGGTACTACGCT (SEQ ID NO: 2)
  • the haploid sperm head of IG-DMR KO mice was injected into the enucleated egg cells by the method of 1.3 above to obtain reconstituted blastocysts, and a solitary male haploid embryonic stem cell line was constructed.
  • a solitary male haploid embryonic stem cell line was constructed.
  • 8 haploid cell lines established in total 2 cell lines were carrying IG-DMR knockouts (named with )
  • the cell was constructed as a donor of ICAMCI to construct a semi-cloned mouse and found Cells were not effective donors for semi-clonal mice (Table 1, Table S1), and it was difficult to obtain healthy normal SC mice (only 4 normal SC mice were obtained from 499 transplanted embryos) (Fig. 2A) , S2E). Moreover, most of the mice are of growth retardation type.
  • H19-3.8K sgRNA-1 CATGAACTCAGAAGAGACTG (SEQ ID NO: 3)
  • H19-3.8K sgRNA-2 AGGTGAGAACCACTGCTGAG (SEQ ID NO: 4)
  • the method by the CRISPR-Cas9 is derived from the foregoing embodiment. ⁇ 3.8 kb DMR knocked out of H19 in cell lines (see Thorvaldsen, JL, Mann, MR, Nwoko, O., Duran, KL, and Bartolomei, MS (2002). Analysis of sequence upstream of the endogenous H19 gene reveals elements Essential and dispensable for imprinting. Molecular and cellular biology 22,2450-2462), and successfully obtained 13 DKO-AG-haESC cell lines (named To ) (Fig. 2C, S2H and Table S2).
  • the oligo of the sgRNA was annealed, and then the sgRNA of H19 and IG-DMR were ligated with the BbsI-digested px330-mCherry plasmid, respectively.
  • the correct bacterial solution was subjected to plasmid extraction for subsequent transfection.
  • Genotype identification method using the method of 1.12 above
  • mice died shortly after birth, which is consistent with the previously reported IG-DMR maternal inheritance that would result in a pre-natal or postnatal death phenotype (Lin, SP, Youngson, N., Takada, S., Seitz, H., Reik, W., Paulsen, M., Cavaille, J., and Ferguson-Smith, AC (2003).
  • RNA-seq and gene expression analysis were performed using the methods of 1.10 and 1.7, respectively.
  • Gapdh-F CACTCTTCCACCTTCGATGC
  • Gapdh-R CTCTTGCTCAGTGTCCTTGC
  • Igf2-F CTAAGACTTGGATCCCAGAACC
  • Igf2-R GTTCTTCTCCTTGGGTTCTTTC
  • Gtl2-F TTGCACATTTCCTGTGGGAC
  • Gtl2-R AAGACACCATAGGCCACTAGG
  • H19-F CATGTCTGGGCCTTTGAA
  • H19-R TTGGCTCCAGGATGATGT
  • Mutation target Tet1, Tet2, Tet3, p53 family mutation
  • the sgRNAs of Tet1, Tet2, and Tet3 were annealed, respectively, and then ligated to the BxsI-cut px330-mCherry On the granules, positive plasmids ligated into sgRNA were picked by sequencing.
  • Tet1 sgRNA sequence GGCTGCTGTCAGGGAGCTCA (SEQ ID NO: 5)
  • Tet2 sgRNA sequence GAAAGTGCCAACAGATATCC (SEQ ID NO: 6)
  • Tet3 sgRNA sequence AAGGAGGGGAAGAGTTCTCG (SEQ ID NO: 7)
  • the plasmids expressing the sgRNAs of Tet1, Tet2, and Tet3 were co-transfected into the DKO-AG-haESCs cell line, and mCherry-positive cells were sorted and plated in a Petri dish. After 5 days of growth, clones were picked and passaged for amplification. The established cell lines were identified by PCR product sequencing to identify mutations in Tet1, Tet2, and Tet3.
  • Tet1check-F GCCCCTGTTGTCTTATACGTT
  • Tet3check-F CAGAGTGGCCTCAGTTTCCC
  • the sgRNAs of p53, p63, and p73 were annealed separately, and then ligated to the BxsI-cut px330-mCherry plasmid, respectively, and the positive plasmid ligated into sgRNA was picked out by sequencing.
  • P53 sgRNA sequence CACCTGGGCTTCCTGCAGTC (SEQ ID NO: 8)
  • P73 sgRNA sequence TGTCGATAGGAGTCAACCAA (SEQ ID NO: 10)
  • the plasmids expressing the sgRNAs of p53, p63, and p73 were co-transfected into the DKO-AG-haESCs cell line, and mCherry-positive cells were sorted and plated in a petri dish. After 5 days of growth, clones were picked and passaged for amplification. The established cell lines were identified by PCR product sequencing to identify mutations in p53, p63, and p73.
  • mice were constructed using the previously constructed cells as donors of ICAMCI using the method of 1.9 above.
  • This example mutates Tet1, Tet2 and Tet3 in DKO-AG-haESCs by the method of CRISPR-Cas9.
  • Cas9 and Tet1, 2, and 3 sgRNAs will be constructed (Fig. 3A) (see Wang, H., Yang, H., Shivalila, CS, Dawlaty, MM, Cheng, AW, Zhang, F., and Jaenisch, R. (2013).
  • Cell 153, 910-918) plasmids were transferred into DKO-AG-haESCs, and finally 56 DKO-AGs were established.
  • sgRNAs of Tet1 and Tet3 were annealed separately, and then ligated into the BbsI-digested px330-mCherry plasmid, and the positive plasmid ligated into sgRNA was picked out by sequencing.
  • pEGFP-N1 plasmid and pECFP-N1 plasmid were used as templates, respectively (primer: P2A-fluorescence F: GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCGAGGAG)
  • P2A-fluorescence R CTTGTACAGCTCGTCCATG
  • the homology arm sequence is as follows:
  • TET1 LA left homology arm: (SEQ ID NO: 11)
  • TET1 RA right homology arm: (SEQ ID NO: 12)
  • TET3 LA left homology arm: (SEQ ID NO: 13)
  • TET3 RA right homology arm: (SEQ ID NO: 14)
  • Tet1 and Tet3 were co-transfected into the DKO-AG-haESCs cell line together with the Tet1-EGFP and Tet3-ECFP donors, and mCherry-positive cells were sorted and plated in a Petri dish. After 5 days of growth, clones were picked and passaged for amplification. The established cell lines were identified by PCR for the knock-in of Tet1-EGFP and Tet3-ECFP. The double knock-in cells were named Tet1&3-KI-DAH.
  • PCR identification primers The following are PCR identification primers:
  • Tet1 LA-F TTTGTGTCTATGAACTACCAGTGAG
  • Tet1 LA-F CAGGCCCGGGGTTTTCTTC
  • Tet1 RA-F CAACGAGAAGCGCGATCACA
  • Tet1 RA-F TTTTGACTGATCCCAATTTGCCT
  • Tet3 LA-F TGTTCACTGGTGAAGGCCAG
  • Tet3 RA-F TGAGCAAAGACCCCAACGAG
  • Tet3 RA-R ATCGACAAACTTTGGGGCGA
  • Tet2 sgRNA was annealed and then ligated into the BbsI-digested px330-mCherry plasmid, and the positive plasmid ligated into the sgRNA was picked out by sequencing.
  • P2A-fluorescence F GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCGAGGAG
  • P2A-fluorescence R CTTGTACAGCTCGTCCATG was used to amplify the sequence encoding mCherry, and then ligated into the pMD19-T vector. Subsequently, the left and right homology arms of the gene of interest were inserted correspondingly on both sides of the mCherry sequence on the vector of pMD 19-T-mCherry.
  • TET2 LA left homology arm: (SEQ ID NO: 15)
  • TET2 RA right homology arm: (SEQ ID NO: 16)
  • Tet2 sgRNA plasmid and the Tet2-mCherry donor were co-transfected into the Tet1&3-KI-DAH cell line, and mCherry-positive cells were sorted and plated in a Petri dish. After 5 days of growth, clones were picked and passaged for amplification. The established cell line was identified by PCR to identify the knock-in of Tet2-mCherry. The positive cell clone is the Tet-TKI-DAH cell line.
  • Tet2 LA-F CACACCCTTCACCAACAGACG
  • Tet2 LA-R ATCTCGAACTCGTGGCCGTT
  • Tet2 RA-F AAGACCACCTACAAGGCCAAG
  • Tet2 RA-R GGTAGGCAAAGTGCTTTTCTAAGAC
  • mice were constructed using the previously constructed cells as donors of ICAMCI using the method of 1.9 above.
  • This example obtained DKO-AG-haESCs in which endogenous Tet1, Tet12 and Tet3 were knocked into different fluorescent reporter groups.
  • the DKO-AG-haESCs were transfected into a double-stranded DNA donor vector in which both Cas9 and Tet1, Tet3sgRNA (Fig. 3A) were expressed, and EGFP and ECFP reporter groups were fused to the last terminator of Tet1 and Tet3, respectively. 3H and S5A).
  • a total of 150 DKO-AG-haESCs cell lines obtained 10 Tet1-EGFP knock-in and 7 Tet3-ECFP knock-in cell lines, respectively ( Figures S5B and S5C).
  • Tet1&3-KI-DAH-1 Tet1&3-KI-DAH-1
  • ICAHCI results showed that DKO-AG-haESCs carrying Tet1-EGFP and Tet3-ECFP knock-in had similar half-clonal mouse birth abilities as those of WT DKO-AG-haESCs ( Figures S5F, S5G, Table 1 and Table S3).
  • Tet-TKI-DAH-1to Tet-TKI-DAH-8 Fig. 3I and S5H.
  • the developmental potential of the Tet-TKI-DAH cell line was verified by the ICAMCI method. Consistently, Tet-TKI-DAH can be efficiently obtained by injection into an egg.
  • the virus preparation was carried out by the method of 1.5 above and the DKO-AG-haESCs were infected with the whole genome sgRNA lentivirus library, the pX330-mCherry plasmid expressing Cas9 was transfected, and finally DKO-AG-haESCs carrying the sgRNA library were obtained.
  • mice were constructed using the previously constructed cells as donors of ICAMCI using the method of 1.9 above.
  • sgRNA The detection of sgRNA is PCR amplification by specific primers, and then running on agarose gel to see if there is a band.
  • Lenti-sg-F GTTACTCGAGCCAAGGTCGG
  • the corresponding gene was inserted according to sgRNA sequencing, and then the upstream and downstream primers were designed by a conventional method in the vicinity of the target gene sgRNA, PCR, and then sequenced.
  • This experiment used a mouse lentivirus library that was just established and clear.
  • This library designed 87,897 sgRNAs for the genes encoding 19,150 mouse-encoded proteins.
  • 1.0 ⁇ 10 7 FACS-enriched haploid cell line Infection was performed using a whole genome sgRNA lentiviral library. After 2 days, these infected cells were treated with puromycin for 7 days, followed by transfection of the pX330-mCherry plasmid expressing Cas9. Haploid cells expressing mCherry indicated successful transfection expression of Cas9, which was used for ICAMCI experiments after FACS enrichment (Fig. 4B).
  • haploid cell clones were randomly picked. All of the cell clones tested carried an sgRNA, and DNA sequencing revealed that these genes of interest were mutated, indicating that the haploid cells were successfully genetically induced by CRISPR-Cas9.
  • DKO-AG-haESCs were infected with a lentiviral library carrying CRISPR-sgRNA using a method of 1.5 to obtain a cell line carrying lentiviral integration of the sgRNA library.
  • the nucleus of the haploid carrying the sgRNA was injected into the mature egg by ICAPCI, and then Cas9 mRNA was injected into the reconstituted egg by cytoplasm injection (this scheme was named: Lenti-sgRNA + Cas9 injection).
  • the semi-cloned mice were then constructed by embryo transfer.
  • the haploid cells carrying sgRNA were constructed by the method of Example 10A, and the pX330-mCherry plasmid was transiently transferred into haploid cells carrying sgRNA.
  • the nucleus of the haploid cells obtained in A was injected into the egg by ICAMCI, followed by injection of Cas9 mRNA by cytoplasmic injection, and then the embryo was transplanted to construct a semi-cloned mouse.
  • the pX330-mCherry plasmid was transiently transferred into haploid cells carrying sgRNA, and injected into the egg followed by Cas9 mRNA (Fig. S6A and S6B) (this scheme was named: Lenti-sgRNA + pX330 + Cas9 injection).
  • Fig. S6A and S6B this scheme was named: Lenti-sgRNA + pX330 + Cas9 injection.
  • a total of 27 SC mice carrying one sgRNA were obtained, 22 carrying gene mutations (Fig. S6C, S6D and Table 2), and 13 of the mutant mice carried biallelic mutations (Fig. S6E) (accounting for all 41.9% of SC mice, and the other 9 mice were single allele mutations.
  • TA cloning and sequencing of 5 biallelic mutant mice showed that about 79% of the clones had insertion deletion mutations ( Figures S6F, S6G, and Table S4).
  • a specific procedure for constructing a cell line using the Lenti-Cas9+lenti-sgRNA method prepared by the method of "Cas9 lentivirus and sgRNA library lentiviral double infection" in 1.5.
  • the Cas9 lentivirus and sgRNA library lentiviral-integrated cell lines were further subjected to flow sorting to enrich the haploid for subsequent ICAMCI manipulation.
  • DKO-AG-haESCs with continuous expression of Cas9 and sgRNA libraries were used as donors of ICAMCI to construct semi-cloned mice using the method of 1.9 above.
  • mice Examples of primers for identifying mutations in mice:
  • Scube1 check-F CCATAATAATCCACTTCCAT
  • AG-haESCs from the prior art (Yang, H., Shi, L., Wang, BA, Liang, D., Zhong, C., Liu, W., Nie, Y., Liu, J. ,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149,605-617) is a gift from the author of the literature
  • AG-haESCs from the prior art (Yang, H., Shi, L., Wang, BA, Liang, D., Zhong, C., Liu, W., Nie, Y., Liu, J. ,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617)

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Abstract

一种孤雄单倍体胚胎干细胞，所述孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR被敲除。所述孤雄单倍体胚胎干细胞的制备方法，以及所述孤雄单倍体胚胎干细胞在构建基因改造半克隆动物及基因改造半克隆动物文库中的用途。所述孤雄单倍体胚胎干细胞能够获得球形精子类似的特性，注射到卵子中能稳定地获得存活的SC小鼠，且能有效地用于多基因遗传操作，进一步得到携带多基因遗传修饰的动物。

Description

一种孤雄单倍体胚胎干细胞及其制备与应用 技术领域
本发明涉及生物技术，特别是涉及一种孤雄单倍体胚胎干细胞及其制备与应用。
背景技术
全基因组水平的隐性基因筛选是鉴定基因在特定生物学过程所起功能的一种极其有效和强大的方法。这种策略已经成功地在低等生物比如酿酒酵母、线虫得以运用。在哺乳动物中，由于基因组的二倍体性，这种功能筛选显得异常的困难(Shi,L.,Yang,H.,and Li,J.(2012).Haploid embryonic stem cells:an ideal tool for mammalian genetic analyses.Protein&cell 3,806-810)。通过靶向mRNA水平的RNA干扰(RNAi)手段目前已经成为哺乳类细胞全基因组范围内进行基因功能丧失筛选的最佳方案。然而，这种方法常常无法有效抑制基因的表达，同时还存在着脱靶效应(Kaelin,W.G.,Jr.(2012).Molecular biology.Use and abuse of RNAi to study mammalian gene function.Science 337,421-422)。最近，来源于细菌的CRISPR-Cas9系统已经成功运用于小鼠和人细胞水平的基因功能缺失的筛选中。然而，CRISPR-Cas9介导的全基因组水平筛选仅使用于细胞水平，而将研究只局限于细胞水平的表型分析。为此，就如同目前在低等生物如酵母线虫所做的那样，如何在哺乳动物系统中实现对范围更广的生物学过程进行高效、大规模、功能缺失的筛选途径。
哺乳动物单倍体胚胎干细胞(haESCs)的获得(Elling,U.,Taubenschmid,J.,Wirnsberger,G.,O'Malley,R.,Demers,S.P.,Vanhaelen,Q.,Shukalyuk,A.I.,Schmauss,G.,Schramek,D.,Schnuetgen,F.,et al.(2011).Forward and reverse genetics through derivation of haploid mouse embryonic stem cells.Cell Stem Cell 9,563-574；Leeb,M.,and Wutz,A.(2011).Derivation of haploid embryonic stem cells from mouse embryos.Nature 479,131-134)，为遗传学分析提供了一种很理想的工具。孤雄单倍体胚胎干细胞(AG-haESCs)的整个基因组是来源于精子，其能够通过注射到成熟的MII卵子(intracytoplasmic AG-haESCs injection,ICAHCI)的方式实现重构胚胎的全程发育，进而获得存活的动物个体，我们将其称为半克隆动物。我们推断，如果孤雄单倍体胚胎干细胞能够通过ICAHCI高效稳定地产生半克隆小鼠的话，那么其作为受精的媒介携带基因组水平的CRISPR-Cas9敲除文库，就能筛选出参与特定发育过程的重要基因。
然而，之前的研究显示出生存活的半克隆小鼠效率非常低(半克隆小鼠的出生效率为4.5％，半克隆大鼠的为1％)，同时有大约50％的半克隆小鼠呈现出发育阻滞的表型，并且出生后不久就死去。更者，随着AG-haESCs长期的培养，半克隆小鼠的整体出生率出现急速下降，特别是进行基因操作所导致的额外培养过程。
其中一个可能的原因是印记基因的异常表达。这些以双亲来源特异表达方式的印记基因，被认为单亲胚胎发育的一个重要障碍，因此胚胎的正常生长和发育同时需要母本和父本的基因组。目前，大约有150个小鼠印记基因已经被鉴定出来。大部分都位于一个很大的基因簇中，并且通过差异性甲基化区域(DMR)进行调控(Bartolomei,M.S.(2009).Genomic imprinting:employing and avoiding epigenetic processes.Genes&development 23,2124-2133)。和这一假说 一致，有研究表明，父源表达抑制的印记基因H19，其所在差异性甲基化区域(DMR)在孤雄单倍体胚胎干细胞和生长阻滞的半克隆小鼠中均一致地发生了印记的异常擦除(Yang,H.,Shi,L.,Wang,B.A.,Liang,D.,Zhong,C.,Liu,W.,Nie,Y.,Liu,J.,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617)。母源表达的H19基因与父源表达的Igf2相邻，它们被同一个DMR调控着，这个DMR在父本等位基因中发生DNA甲基化，并且作为CTCF依赖的绝缘子只允许母源等位基因进行表达。这一DMR的甲基化状态决定着H19(H19表达，DMR去甲基化，绝缘子活化)和Igf2(Igf2表达，DMR甲基化，绝缘子失活)是否表达。有趣的是，H19基因或者其DMR的敲除对于小鼠来说并没有任何严重的表型(Leighton,P.A.,Ingram,R.S.,Eggenschwiler,J.,Efstratiadis,A.,and Tilghman,S.M.(1995).Disruption of imprinting caused by deletion of the H19gene region in mice.Nature 375,34-39；Thorvaldsen,J.L.,Mann,M.R.,Nwoko,O.,Duran,K.L.,and Bartolomei,M.S.(2002).Analysis of sequence upstream of the endogenous H19gene reveals elements both essential and dispensable for imprinting.Molecular and cellular biology 22,2450-2462)。目前，有关于采用完全成熟的卵子和基因改造的未成熟卵子获得的重构胚的相关研究(Kono,T.,Obata,Y.,Wu,Q.,Niwa,K.,Ono,Y.,Yamamoto,Y.,Park,E.S.,Seo,J.S.,and Ogawa,H.(2004).Birth of parthenogenetic mice that can develop to adulthood.Nature 428,860-864；Kawahara,M.,Wu,Q.,Takahashi,N.,Morita,S.,Yamada,K.,Ito,M.,Ferguson-Smith,A.C.,and Kono,T.(2007).High-frequency generation of viable mice from engineered bi-maternal embryos.Nature biotechnology 25,1045-1050)。然而未成熟的卵子由于不能在体外培养扩增，因此导致其无法在体外完成基因改造，并且由于完全成熟的卵子和基因改造的未成熟卵子获得重构胚的技术操作难度高，所以应用价值不大。
发明内容
鉴于现有技术半克隆动物出生率低下的缺点，本发明的目的在于提供一种可提高半克隆动物出生率的孤雄单倍体胚胎干细胞及其应用。
导致出生率极有可能的原因是孤雄单倍体本身的印记状态出现了异常。通过实验，惊讶的发现，将H19-Igf2和Dlk1-Dio3两个印记簇的差异性甲基化区域(DMR)敲除可以重新建立起孤雄单倍体胚胎干细胞球形精子相类似的特性。将这种两个DMR敲除的孤雄单倍体胚胎干细胞命名为DKO-AG-haESCs。这种DKO-AG-haESCs能够高效地获得半克隆小鼠(大约有20％的SC小鼠出生率)，并且通过在体外对孤雄单倍体胚胎干细胞进行基因操作后仍能够稳定地产生多基因修饰的半克隆动物。更让人兴奋的是，这种DKO-AG-haESCs只需要在体外细胞水平上转染稳定表达的sgRNA文库和Cas9就能够有效地一步法获得大量的突变动物。这些实验结果表明DKO-AG-haESCs可以作为一个中间体通过携带sgRNA文库的方式在个体水平上实现基因的突变，因此可进一步运用到动物个体水平上基于基因突变的大规模筛选上。
本发明第一方面提供了一种孤雄单倍体胚胎干细胞，所述孤雄单倍体胚胎干细胞的H19DMR及IG-DMR基因被敲除。
本发明第二方面提供了所述孤雄单倍体胚胎干细胞的制备方法，包括将孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR敲除获得所述孤雄单倍体胚胎干细胞。
本发明第三方面提供了所述孤雄单倍体胚胎干细胞的用途，为用于构建基因改造的半克 隆动物。
本发明第四方面提供了一种构建基因改造半克隆动物的方法，将H19 DMR及IG-DMR双敲除孤雄单倍体胚胎干细胞与卵细胞结合获得半克隆胚胎，培育所述半克隆胚胎获得半克隆动物。
本发明第五方面提供了一种基因改造动物，由前述方法构建获得，或者为前述方法构建的半克隆动物的有性繁殖后代。
本发明第六方面提供了一种本发明的孤雄单倍体胚胎干细胞及sgRNA慢病毒文库构建基因改造半克隆动物文库的方法。
本发明第七方面提供了一种基因改造半克隆动物文库，为采用前述方法构建获得。
本发明的有益效果：
(1)、现有的研究表明：从单倍体囊胚建立的AG-haESCs能够通过ICAHCI技术获得半克隆动物。然而AG-haESCs经过长期的培养传代特别是体外基因操作之后无法产生存活的SC动物，可能的原因是是由于雄性印记的丢失。本发明发现通过敲除H19和IG-DMR的敲除AG-haESCs能够获得球形精子类似的特性。AG-haESCs注射到卵子中能够以约20％的效率稳定地获得存活的SC小鼠，是早期代数WT AG-haESCs效率的约10倍高(Yang et al.,2012)。重要的是，本发明还发现DKO-AG-haESCs能有效地用于多基因遗传操作，进一步通过ICAHCI得到携带多基因遗传修饰的SC小鼠。更者，结合sgRNA文库，DKO-AG-haESCs能高效地用于一步产生杂合和纯合的突变小鼠，为在动物个体水平上实现基于基因敲除的筛选提供进一步的证据。
(2)、DKO-AG-haESCs相比目前已有的获得基因修饰动物的方法有几方面的优势。首先，目前的研究报道了直接注射Cas9mRNA和sgRNA到受精卵一步法获得转基因或内源基因突变的小鼠。这一方法展示了CRISPR-Cas9技术在制备基因修饰动物上强大的潜力。然而，目前观察到的基因操作动物存在极高比率的嵌合现象，这大大地增加了F1代动物表型分析的难度。相比，DKO-AG-haESCs介导的基因编辑技术提供了一套独特的系统，基因修饰的DKO-AG-haESCs可能得到很好的分析和筛选，以确保产生的SC动物能够出现预期的遗传学形状，这样就能大大避免嵌合现象的发生。第二，用传统的二倍体胚胎干细胞注射到囊胚中获得基因修饰的小鼠需要通过嵌合小鼠的生殖系传递。然而，生殖系传递这一步往往非常耗时。更严重的是，针对多基因修饰的二倍体胚胎干细胞，在生殖系传递的过程中会出现后代发生性状分离的现象，只有一小部分的后代可能囊括了所有想要的基因修饰。而DKO-AG-haESCs能够实现完全一步获得多基因修饰的F0小鼠。第三，通过将二倍体胚胎干细胞注射到四倍体囊胚(也称四倍体补偿技术)的方法能够一步得到全部ESC来源的小鼠。这一方法也被广泛使用，是检验重编程细胞例如诱导多能性胚胎干细胞(iPSCs)和核移植胚胎干细胞(ntESCs)多能性的最严格标准。然而，胚胎干细胞或诱导多能胚胎干细胞来源的动物出生效率非常低，可能是由于二倍体胚胎干细胞表观遗传的不稳定性。而DKO-AG-haESCs经过长期的体外培养特别是遗传操作后仍能稳定高效地获得。第四，ICAHCI技术和ROSI相似，ROSI这一项技术已经在不同的哺乳动物中很成熟，包括非人灵长类和人。目前食蟹猴来源的单倍体胚胎干细胞系已经成功建立，ICAHCI技术或许在不久的将来可以用于高效快速地获得基因修饰的非人灵长类动物。最后，DKO-AG-haESCs可以通过sgRNA 文库获得大量基因突变的动物。很显然，针对大规模获得突变动物，这种方法相比直接往胚胎里注射质粒或mRNA的方法更加简单省力，因为携带持续表达Cas9和sgRNA文库的DKO-AG-haESCs系可以反复作为注射的供体，以持续高效地产生不同的基因突变动物。这些不同的突变动物可以简单快速地通过一份通用引物PCR鉴定sgRNA的插入(就像是突变动物的条形码)。相反，通过直接往胚胎注射质粒或mRNA的方法需要将每次独立注射的sgRNA单独分开准备。获得的突变动物需要分开养殖，后续的鉴定需要针对特定的基因使用相应的引物。可以设想，针对适当的sgRNA文库，例如通过细胞水平高通量分析所得到的一些差异表达基因，可以做成一个sgRNA的亚文库，或许这些基因将参与到发育过程的某个特定阶段。利用DKO-AG-haESCs这一方案，可以快速有效地在个体水平上筛选出与这发育过程相关的重要基因。
(3)、DKO-AG-haESCs介导SC动物的获得，是产生携带有基因家族里多基因突变或多个基因敲入等基因修饰动物模型的一种有效而简单的方法。更者，结合sgRNA文库，DKO-AG-haESCs能够一步获得大规模的基因突变动物。虽然DKO-AG-haESCs能如此高效获得SC动物存在的机制仍让人好奇，但是这种方法或许能够促进我们对发育学过程和复杂疾病进行更深入的探究。
附图说明
图1A携带2个DMR敲除的AG-haESCs卵胞浆内注射(ICAHCI)获得SC小鼠的示意图。
图1B
Figure PCTCN2015083165-appb-000001
精子注射到去核MII卵获得孤雄单倍体胚胎。
图1C孤雄单倍体囊胚建立的其中一株孤雄单倍体胚胎干细胞。
图1D经过多次流式富集单倍体后建立的
Figure PCTCN2015083165-appb-000002
孤雄单倍体胚胎干细胞
Figure PCTCN2015083165-appb-000003
图1E
Figure PCTCN2015083165-appb-000004
(p8)通过ICAHCI获得SC小鼠。
图1F敲除IG-DMR的sgRNA序列示意图
图1G获得
Figure PCTCN2015083165-appb-000005
细胞系。左侧：收集mCherry阳性的细胞铺至培养板中。右侧：建立好的
Figure PCTCN2015083165-appb-000006
细胞系
Figure PCTCN2015083165-appb-000007
图1H
Figure PCTCN2015083165-appb-000008
(p29)通过ICAHCI获得SC小鼠。
图S1A
Figure PCTCN2015083165-appb-000009
细胞系的基因型鉴定。
图S1B
Figure PCTCN2015083165-appb-000010
细胞重构的胚胎有80％以上能达到2-细胞，和球形精子注射(ROSI)的发育效率相似。
图S1C
Figure PCTCN2015083165-appb-000011
2、3细胞系通过ICAHCI获得SC小鼠。星号代表出生阻滞的SC小鼠。
图S1D对
Figure PCTCN2015083165-appb-000012
细胞来源的SC小鼠进行基因型鉴定。
图S1E
Figure PCTCN2015083165-appb-000013
细胞来源的正常和阻滞型SC小鼠对其不同的器官进行印记基因(Gtl2和Dlk1)的表达分析。
图S1F在生长阻滞型SC小鼠中，IG-DMR的印记甲基化出现严重的擦除。
图S1G生长阻滞和正常的SC小鼠IG-DMR的Cobra分析。
图S1H
Figure PCTCN2015083165-appb-000014
细胞系的IG-DMR甲基化状态。
图2A
Figure PCTCN2015083165-appb-000015
细胞系(p17)获得的半克隆小鼠，星号代表生长阻滞型小鼠，出生后不久会死。
图2B
Figure PCTCN2015083165-appb-000016
细胞系获得的正常SC小鼠和异常SC小鼠的H19 DMR甲基化状态。
图2C H19 DMR敲除的sgRNAs设计示意图。
图2D所获得的
Figure PCTCN2015083165-appb-000017
细胞系基因型鉴定，这些细胞系是在AGH-OG3细胞系上敲除H19和IG-DMRs所获得。
图2E DKO-AG-haESCs获得的SC小鼠交配后所产生后代的基因型鉴定，只要携带IG-DMR的后代在出生前或出生后不久便会死去。
图2F通过RNA-seq分析DKO-AG-haESCs的基因表达谱。基因的表达谱是将所有基因的表达进行聚类。这三株DKO-AG-haESCs细胞系表现出和对照组AG-haESCs相似的基因表达谱，但是与球形精子极其不同。为了避免单倍体二倍体化对表达谱的影响，我们均通过FACS收集G0/G1的细胞进行RNA-seq。
图2G DKO-AG-haESCs的印记基因表达谱。三株不同的DKO-AG-haESCs表达谱与对照组AG-haESCs相似，但与小鼠的球形精子不同。
图2H通过RRBS分析DKO-AG-haESCs的甲基化情况。
图S2A对
Figure PCTCN2015083165-appb-000018
细胞敲除IG-DMR所获得的
Figure PCTCN2015083165-appb-000019
细胞系进行基因型鉴定。
图S2B DKO-AG-haESCs细胞系
Figure PCTCN2015083165-appb-000020
的测序结果。
图S2C
Figure PCTCN2015083165-appb-000021
细胞ICAHCI获得的2-细胞胚胎。
图S2D SC小鼠的基因型鉴定。
Figure PCTCN2015083165-appb-000022
来源的SC小鼠均存在H19和IG DMR敲除。
图S2E
Figure PCTCN2015083165-appb-000023
(p8)获得的SC小鼠。
图S2F
Figure PCTCN2015083165-appb-000024
细胞的H19 DMR甲基化状态。
图S2G
Figure PCTCN2015083165-appb-000025
得到的正常和生长阻滞型SC小鼠其H19 DM的R甲基化状态。
图S2H DKO-AG-haESCs细胞系
Figure PCTCN2015083165-appb-000026
的测序分析。
图S2I
Figure PCTCN2015083165-appb-000027
细胞(p24)ICAHCI获得SC小鼠。
图S2J
Figure PCTCN2015083165-appb-000028
来源的SC小鼠进行基因型鉴定。
图3A Tet1、Tet12和Tet13sgRNA示意图
图3B FACS收集mCherry阳性的细胞，铺到培养皿中以获得Tet家族基因突变的DKO-AG-haESCs
图3C获得Tet-TKO-DAH细胞系。
图3D Tet-TKO-DAH细胞系中Tet1、Tet2和Tet3的测序。
图3E Tet-TKO-DAH-1和Tet-TKO-DAH-2细胞系获得SC小鼠进行Tet1、Tet2和Tet3基因的PCR产物测序。
图3F p53、p63和p73sgRNA示意图。
图3G p53-TKO-DAH-2细胞系中p53、p63和p73测序。
图3H Tet1-EGFP、Tet2-mCherry、Tet1-ECFP双链DNA载体的示意图。EGFP、mCherry、ECFP分别融合到Tet1、Tet12和Tet13的终止密码子处。
图3I Tet-TKO-DAH-1细胞系的基因型鉴定。
图S3A在WT-AG-haESCs(AGH-OG3)上敲除H19和IG-DMR获得的2株DKO-AG-haESCs细胞系(
Figure PCTCN2015083165-appb-000029
和
Figure PCTCN2015083165-appb-000030
)测序分析。
图S3B
Figure PCTCN2015083165-appb-000031
(p26)通过ICAHCI获得SC小鼠。
图S3C对
Figure PCTCN2015083165-appb-000032
细胞系获得的SC小鼠进行基因型鉴定。
图S3D DKO-AG-haESCs和正常AG-haESCs印记基因(H19,Igf2,Gtl2和Dlk1)的表达分析。H19和IG-DMR敲除后，AGH-OG-3的H19和Gtl2表达水平出现下调。相反，Igf2和Dlk1的表达水平则出现上调。
图S3E H19和Gtl2的bigwig track。RNA-seq结果显示：相比于WT-AG-haESCs，DKO-AG-haESCs的H19和Gtl2表达水平要更低。
图S3F H19-Igf2和Dlk1-Dio3印记簇区域的甲基化水平。
图S3G DKO-AG-haESCs、WT-AG-haESCs和球形精子的印记基因甲基化状态。
图4A携带sgRNA文库的DKO-AG-haESCs经ICAHCI获得大量杂合突变SC小鼠的示意图。
图4B代表Cas9成功转入表达的mCherry阳性单倍体用FACS的方法进行富集，并用于后续的ICAHCI。
图4C对单细胞来源的单倍体克隆进行sgRNA PCR鉴定。所有检测的细胞克隆均带有sgRNA.
图4D对细胞克隆中不同的突变基因进行测序。检测的克隆均存在目的基因的修饰。
图4E来源于携带sgRNA文库DKO-AG-haESCs的SC小鼠。
图4F SC小鼠sgRNA的PCR鉴定。
图4G SC小鼠不同目的基因的突变测序。所有检测的SC小鼠均存在目的基因的修饰。
图S4A Tet-TKO-DAH-3细胞系Tet1、Tet12和Tet13的测序。
图S4B Tet-TKO-DAH-3(p36)经ICAHCI获得SC小鼠。
图S4C p53-TKO-DAH-1细胞系p53、p63和p73的测序。
图S4D p53-TKO-DAH-1细胞系(p44)ICAHCI获得SC小鼠。
图S4E分别从p53-TKO-DAH-1和p53-TKO-DAH-2细胞系得到SC小鼠对p53、p63和p73基因进行测序。
图5A携带持续表达Cas9和sgRNA文库的DKO-AG-haESCs经ICAHCI获得大量双等位基因突变SC小鼠的示意图。
图5B单细胞来源的细胞克隆Cas9的PCR分析。所有检测的细胞克隆均含有Cas9转基因。
图5C单细胞来源的细胞克隆Cas9的定量PCR分析。
图5D对单细胞来源的细胞克隆进行sgRNA PCR鉴定。
图5E来源于携带持续表达Cas9和sgRNA文库DKO-AG-haESCs的SC小鼠。
图5F PCR鉴定SC小鼠的sgRNA。
图5G携带持续表达Cas9和sgRNA文库的DKO-AG-haESCs经ICAHCI获得双等位基因突变SC小鼠。以Polm双等位基因突变的SC小鼠为例。
图5H通过TA克隆和测序分析Polm基因在鼠尾的突变情况。26个检测的克隆中有24个发生了移码突变。
图5I 7只双等位基因突变SC小鼠的TA克隆测序结果总结。超过80％的克隆发生插入缺失突变。
图5J Scube1双等位基因突变的SC小鼠对其不同的器官进行TA克隆和测序分析。
图S5A Tet1-EGFP、Tet2-mCherry和Tet3-ECFP外源双链载体的示意图。
图S5B Tet1-EGFP敲入的DKO-AG-haESCs基因型鉴定。
图S5C Tet3-ECFP敲入的DKO-AG-haESCs基因型鉴定。
图S5D Tet1&3-KI-DAH-1细胞系的基因型鉴定。
图S5E Tet1&3-KI-DAH-1细胞系Tet1-EGFP和Tet-ECFP的测序。
图S5F Tet1&3-KI-DAH-1细胞系(p40)经ICAHCI获得的SC小鼠已长成3周大。
图S5G Tet1&3-KI-DAH-1细胞系获得的SC小鼠基因型鉴定。
图S5H Tet-TKI-DAH-1细胞系中Tet2-mCherry测序。
图S5I Tet-TKI-DAH-2细胞系(p50)获得SC小鼠。
图S5J Tet-TKI-DAH-1细胞系获得SC小鼠的基因型鉴定。
图S6A瞬转pX330-mCherry质粒并携带sgRNA的单倍体细胞注射到成熟的卵细胞，接着向重构的卵细胞注射Cas9mRNA(此方案命名：Lenti-sgRNA+pX330+Cas9injection)。
图S6B单细胞来源的单倍体ES克隆PCR鉴定其sgRNA插入。
图S6C携带sgRNA文库的DKO-AG-haESCs获得SC小鼠。
图S6D SC小鼠的sgRNA PCR鉴定。
图S6E携带sgRNA文库的DKO-AG-haESCs注射到卵细胞，接着向SC胚胎中注射Cas9mRNA。以携带Slco5a1基因双等位基因突变的SC小鼠为例。
图S6F Slco5a1基因的鼠尾TA克隆和测序分析。检测的20个克隆里有18个发生插入缺失突变。
图S6G 4只双等位基因突变小鼠TA克隆和测序的总结分析。
具体实施方式
本发明首先提供了一种孤雄单倍体胚胎干细胞，所述孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR被敲除。
所述孤雄单倍体胚胎干细胞的整个基因组来源于精子，具有干细胞的自我复制能力与多 能性，能取代精子与卵母细胞结合支持胚胎的完全发育。
所述H19 DMR是指：H19-Igf2印记簇内的一段甲基化差异区域(DMR：differentially methylated region)。H19 DMR的具体位置与序列可根据现有的甲基化测序或同源序列分析预测等方法明确。已知人H19 DMR定位于染色体11p15.5区，鼠H19 DMR则位于7号染色体远端，位于H19和Igf2两个基因之间，H19基因的上游的2kb到4kb位置。H19 DMR在父本等位基因上呈甲基化状态，导致CTCF蛋白无法结合到这段甲基化区域上，使H19下游的增强子不需要逾越CTCF这一障碍，进而达到增强上游的Igf2的表达并且降低H19的表达。而在母本等位基因上呈去甲基化状态，CTCF蛋白能够结合到这段未甲基化的区域，因此H19下游的增强子只能增强H19的表达，但是对上游的Igf2无法调控。如果将父本H19DMR敲除的话，那么H19下游的增强子就能上调Igf2的表达。由于孤雄单倍体是父本来源，理论上应当处于完全甲基化的状态，但是研究发现，体外培养的孤雄单倍体H19 DMR的甲基化出现异常擦除，成为去甲基化状态，导致H19的表达异常上调而Igf2的表达被下调，本发明敲除了H19 DMR，纠正了这种H19表达上调Igf2表达下调的异常状态。
所述IG-DMR是指：Dlk-Dio3印记簇内的一段甲基化差异区域(DMR：differentially methylated region)。IG DMR的具体位置与序列可根据现有的甲基化测序或同源序列分析预测等方法明确。已知小鼠的IG-DMR位于12号染色体上，处在印记簇中Dlk1和Gtl2 2个基因之间4.15kb的一段重复序列，人的则位于14号染色体上(14q32.2)。IG-DMR在父本等位基因中，这段区域发生DNA甲基化，这一印记簇的基因Gtl2以及一些mircroRNA不表达，但是基因Rtl1、Dlk1和Dio3则表达。在母本等位基因中，这段区域不发生DNA甲基化(去甲基化状态)，因此Gtl2以及一些mircroRNA进行表达，但是基因Rtl1、Dlk1和Dio3则不表达。在孤雄单倍体(父本来源)和出生异常的SC动物中，研究发现本应该是甲基化状态的IG-DMR，其甲基化出现异常擦除，从而导致基因Rtl1、Dlk1和Dio3的沉默，Gtl2以及一些mircroRNA的异常激活。
进一步的，所述孤雄单倍体胚胎干细胞除了H19 DMR及IG-DMR敲除之外，还经过其他基因改造。
基因改造具体是指通过生物或化学或物理的手段使基因相比改造前在结构上发生变化。这种变化主要是指碱基对组成的变化，包括但不限于一个或多个碱基对的替换、增添、缺失引起的改变。
在一优选例中，列举了对Tet1、Tet2、Tet3、p53家族的基因改造。
所述孤雄单倍体胚胎干细胞来源于哺乳动物，可以为人或非人哺乳动物。较佳的，所述孤雄单倍体胚胎干细胞来源于啮齿动物，如兔、鼠，鼠可以为小鼠、大鼠。在优选的实施例中，所述孤雄单倍体胚胎干细胞来源于小鼠。
相比H19 DMR及IG-DMR未同时敲除的孤雄单倍体胚胎干细胞，本发明的孤雄单倍体胚胎干细胞构建半克隆动物的出生率更高。
本发明还提供了所述孤雄单倍体胚胎干细胞的制备方法，包括将孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR敲除获得所述孤雄单倍体胚胎干细胞。
可以采用现有的基因编辑方法敲除所述H19 DMR及IG-DMR。在优选的实施例中，采用CRISPR/Cas9介导的基因操作敲除H19 DMR及IG-DMR。还可采用其他方法实现基因敲 除，并不限于实施例所列举的方式。
基于H19 DMR的敲除，H19 DMR的完整序列从染色体DNA中去除；基于IG DMR的敲除，IG-DMR的完整序列从染色体DNA中被去除。
在一实施例中，先构建H19 DMR敲除的孤雄单倍体胚胎干细胞，并在此基础上进一步敲除IG-DMR。在另一个实施例中，先构建IG-DMR敲除的孤雄单倍体胚胎干细胞，并在此基础上进一步敲除H19 DMR。在另一个实施例中，直接构建H19 DMR及IG-DMR同时敲除的孤雄单倍体胚胎干细胞。
进一步的，所述孤雄单倍体胚胎干细胞还经其他基因改造。
所述的其他基因改造是指除H19 DMR及IG-DMR双敲除以外的基因改造。所述其他基因改造可以是单个目标基因的改造或者为多个感兴趣目标基因的改造。感兴趣目标基因并不特指，可以根据研究需要设定并改造。例如，可以是一个、两个、三个以上目标基因的改造。由于本发明H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞可在体外传代，因此理论上可以不断对其进行基因改造，对于目标基因的改造数量可以按照需要操作，没有特别限制。
所述基因改造包括但不限于目标基因的敲入、目标基因的敲除等。目标基因的敲入、目标基因的敲除可以采用基因打靶、同源重组等技术来完成，包括但不限于基于ZFN(锌指核酸酶)、TALEN(转录激活样效应因子核酸酶)和CRISPR/Cas9(成簇规律间隔短回文重复技术)等的基因操作。
在一实施例中，将H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞进一步经单次或多次基因改造获得H19 DMR及IG-DMR双敲除且经其他基因改造的孤雄单倍体胚胎干细胞。或者，也可以先对孤雄单倍体胚胎干细胞进行基因改造，而后将基因突变的孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR敲除。除以上列举，凡是可以实现H19 DMR及IG-DMR双敲除及其他基因突变的其他生物技术手段均可用于构建H19 DMR及IG-DMR双敲除且经其他基因改造的孤雄单倍体胚胎干细胞。
本发明还提供了所述孤雄单倍体胚胎干细胞的用途，为用于构建基因改造的半克隆动物。
进一步的，以所述孤雄单倍体胚胎干细胞替代精子作为受精媒介用于构建基因改造的动物。
本发明还提供了一种构建基因改造半克隆动物的方法，将H19 DMR及IG-DMR双敲除孤雄单倍体胚胎干细胞与卵细胞结合获得半克隆胚胎，培育所述半克隆胚胎获得半克隆动物。
一般情况下，所述卵细胞与所述孤雄单倍体胚胎干细胞来源于同类动物，较佳的，为同种动物。
半克隆胚胎具体可以H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎。
进一步的，可采用胚胎移植的方法在合适的母体内培育所述半克隆胚胎获得半克隆动物。
在一优选的实施例中，所述合适的母体选用假孕ICR母鼠。
进一步的，所述孤雄单倍体胚胎干细胞经其他基因改造。
本发明还提供了一种基因改造动物，由前述方法构建获得，或者为前述方法构建的半克隆动物的有性繁殖后代。
本发明所述半克隆动物可以为非人哺乳动物。较佳的，所述半克隆动物为啮齿动物，如 兔、鼠。在优选的实施例中，所述半克隆动物为小鼠。
本发明还提供了构建基因改造半克隆动物文库的方法，包括下列步骤：
1)以sgRNA慢病毒文库质粒制备的病毒颗粒感染本发明的孤雄单倍体胚胎干细胞，获得携带sgRNA文库的孤雄单倍体胚胎干细胞文库；
2)以携带sgRNA文库的孤雄单倍体胚胎干细胞库中的孤雄单倍体胚胎干细胞作为ICAHCI的供体，利用表达Cas9的载体和/或Cas9的mRNA，采用ICAHCI法获得半克隆胚胎文库；
3)培育所述半克隆胚胎文库中的胚胎获得半克隆动物文库。
所述sgRNA慢病毒文库质粒包含数个表达不同sgRNA的慢病毒载体。sgRNA慢病毒文库可以利用现有技术构建，也可采购现有的sgRNA慢病毒文库质粒。具体的，可以将针对不同基因所设计的sgRNA克隆入慢病毒载体获得。所述sgRNA可以根据自己所感兴趣的基因进行设计。
在一优选实施例中，采用的为商购的小鼠全基因组的sgRNA慢病毒文库。
具体的，步骤2)可选自以下任一：
方法A：
向携带sgRNA文库的孤雄单倍体胚胎干细胞文库进一步转染表达Cas9的质粒，将获得的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎。
方法B：
以携带sgRNA文库的孤雄单倍体胚胎干细胞文库的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法注入成熟卵子，再向重构的卵子注入Cas9mRNA获得半克隆胚胎。
方法C：
向携带sgRNA文库的孤雄单倍体胚胎干细胞文库进一步转染表达Cas9的质粒，将获得的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法注入成熟卵子，再向重构的卵子注入Cas9mRNA获得半克隆胚胎，再经胚胎移植获得半克隆动物。
上述方法A和C中所述表达Cas9的质粒可将表达Cas9的基因克隆入表达质粒构建。在一优选实施例中，所述表达Cas9的质粒为pX330-mCherry质粒。可用于构建表达Cas9的质粒并不限于pX330质粒。所述表达质粒只需要适合在哺乳动物细胞中进行外源基因的表达即可。
本发明还提供了另一种构建基因改造半克隆动物文库的方法，包括下列步骤：
1)以表达Cas9的慢病毒颗粒和sgRNA慢病毒文库制备的慢病毒颗粒感染本发明的孤雄单倍体胚胎干细胞，获得携带sgRNA文库以及Cas9持续表达的孤雄单倍体胚胎干细胞文库；
2)以携带sgRNA文库以及Cas9持续表达的孤雄单倍体胚胎干细胞文库中的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎文库；
3)培育所述半克隆胚胎文库中的胚胎获得半克隆动物文库。
所述表达Cas9的病毒颗粒可采用现有技术，将表达Cas9的编码基因克隆入慢病毒载体然后进行慢病毒包装获得，表达Cas9的慢病毒载体可商购获得。
所述半克隆动物文库包含数个基因突变的半克隆动物。所述动物可以是杂合子或双等位 基因突变的突变动物。
进一步的，可采用胚胎移植的方法在合适的母体内培育所述半克隆胚胎获得半克隆动物。
在一优选实施例中，所述合适的母体可选用假孕ICR母鼠。
本发明还提供了一种基因改造半克隆动物文库，为采用前述方法构建获得。
本发明的基因改造半克隆动物文库可在下属个体水平进行基因的遗传筛选。
本发明所述半克隆动物文库可以为非人哺乳动物文库。较佳的，所述半克隆动物文库为啮齿动物文库，如兔文库、鼠文库。在优选的实施例中，所述半克隆动物文库为小鼠文库。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明。
简写：
AG-haESCs：孤雄单倍体胚胎干细胞
DKO-AG-haESCs：H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞
1.实验材料与方法
1.1材料和试剂
细胞培养基(DMEM)、胎牛血清(FBS)、血清替代物(KSR)、胰酶、Opti-MEM、DPBS、Lipofectamine 2000购自Life Technologies公司；限制性内切酶、T4连接酶购自NEB公司；Taq酶和dNTPs购自TaKaRa公司；CDNA反转录试剂盒、荧光定量试剂SYBR-Green购自TOYOBO公司；寡聚核苷酸由上海generay公司合成。
HEPES-CZB培养液：
H-CZB Stock 98.5ml、Hepes.2Na(sigma,CAT#H0763)或ICN 520mg或Hepes(sigma,CAT#H4034)476mg、NHCO3 42mg、CaCl2·2H2O 100×stock 1ml、Pyruvate 3.0mg、Glutamin 200×stock 0.5ml调整pH至7.4，混匀过滤
H-CZB stock:
CZB stock 500ml,PVA(sigma,P8136)50mg、CZB stock:H2O 985ml、NaCl(sigma,CAT#S5886)4760mg、KCL(sigma,CAT#P5405)360mg、MgSO4·7H2O(sigma,CAT#M1880)290mg、EDTA·2Na(sigma,CAT#E6635)40mg、Na-Lactate(sigma,CAT#L7900)5.3ml、D-Glucose(sigma,CAT#G6152)1000mg、KH2PO4(sigma,CAT#P5655)160mg
激活液：10mM Sr2+,5ng/ml Trichostatin A(TSA)
KSOM培养液(KSOM+AA with glucose)：millipore,CAT#MR-106-D
ESC培养基：
DMEM(millipore,CAT#SLM-220-M)75％、20％血清替代物KSR(Gibco,CAT#10828-028)、1,500U/ml LIF(Millopre,CAT#ESG1107)、3M CHIR99021(Stemgent,CAT#04-0004)和1M PD0325901(Stemgent,CAT#04-0006)
Acid Tyrode溶液：sigma,CAT#T1788
CZB培养液：
CZB stock 99ML、CaCl2·2H2O 100×stock 1ml、Pyruvate 3.0mg、Glutamin 200×stock0.5ml、BSA 500mg
pX330-mCherry：
px330(addgene)质粒用NotI进行酶切，然后将从pmCherry-C1(Clontech)上扩增好的CMV-mcherry-pA片段插入进酶切过的px330质粒获得。
扩增所用引物：
mCherry-F:ATTTGCGGCCGCATAGTAATCAATTACGGG
mCherry-R:ATTTGCGGCCGCATGCAGTGAAAAAAATGC
小鼠的sgRNA慢病毒文库：Addgene公司
Cas9的病毒质粒：Addgene公司提供
Cas9的mRNA:
参考Wang,H.,Yang,H.,Shivalila,C.S.,Dawlaty,M.M.,Cheng,A.W.,Zhang,F.,and Jaenisch,R.(2013).One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell 153,910-918.的记载获得
球形精子：
将小鼠睾丸用胶原酶IV消化20min后，用胰酶消化10min,然后通过FACS(流式分选)得到小鼠的球形精子。
1.2试验动物
所有的动物使用流程都是按照中科院上海生命科学研究院生化与细胞所的动物轮流手册。
H19 Δ3.8kb KO小鼠(C57/B6背景,纯合)：参考文献构建(Thorvaldsen,J.L.,Mann,M.R.,Nwoko,O.,Duran,K.L.,and Bartolomei,M.S.(2002).Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting.Molecular and cellular biology 22,2450-2462.)
IG-DMR KO小鼠(C57/B6背景,杂合)：参考文献构建(Lin,S.P.,Youngson,N.,Takada,S.,Seitz,H.,Reik,W.,Paulsen,M.,Cavaille,J.,and Ferguson-Smith,A.C.(2003).Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12.Nature genetics 35,97-102，)
B6D2F1(C57BL/6X DBA2)雌性小鼠：用C57BL/6品系的母鼠和DBA2品系的公鼠交配后得到的雌性后代。
假孕ICR母鼠：ICR成年鼠购于斯莱克，将ICR成年母鼠与结扎的ICR公鼠交配即得到ICR假孕母鼠
1.3孤雄单倍体胚胎干细胞系的建立
按照已报道的方法构建(Yang,H.,Shi,L.,Wang,B.A.,Liang,D.,Zhong,C.,Liu,W.,Nie,Y.,Liu,J.,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617，)。
方法：
去掉MII卵子的细胞核注入相应的精子头。小鼠MII卵在人绒毛膜促性腺激素(HCG)处理的14小时后进行收集，然后利用Piezo针在含有5ug/ml细胞松弛素B(CB)的HEPES-CZB培养液中去核。去核后，单个的精子头注入到卵胞质中。重构的胚胎在CZB培养液中培养1小时，然后转入到含1mM Sr2+的激活液进行激活。激活之后，所有的重构胚胎转到含有氨基酸的KSOM培养液于37℃，5％CO2条件下进行培养。3.5天后到达桑葚或囊胚阶段的重构胚胎种植到ESC培养基中。
重构胚胎透明带由Acid Tyrode溶液进行消化去除。每个转移到铺有小鼠成纤维细胞滋养层的96孔板孔中，并且用含有20％血清替代物(KSR)、1,500U/ml LIF、3M CHIR99021和1M PD0325901的ESC培养基进行培养。培养4-5天后，细胞克隆由胰酶消化并传到铺有新鲜滋养层的96孔板中。细胞进一步扩大培养，传代到48孔板并进一步至6孔板中，日常的细胞维持仅在6孔板中即可。为了分选出单倍体细胞，胚胎干细胞胰酶消化后，用PBS(GIBCO)洗一遍，然后在含有15μg/ml Hoechst 33342的ESC培养基。水浴30min。随后，通过流式分选仪BD FACS AriaII分选收集出单倍体1N峰形的细胞进行后续的培养获得孤雄单倍体胚胎干细胞系。
1.4CRISPR-Cas9介导的基因操作
CRISPR-Cas9质粒构建：将合成好的sgRNA的正向寡核苷酸链和反向寡核苷酸链进行退火获得双链寡核苷酸链(本发明中sgRNA序列均指sgRNA的正向寡核苷酸链序列)然后与BbsI(New England Biolabs)酶切好的pX330-mCherry进行连接。构建好的相应质粒按照说明书用Lipofectamine 2000(Life Technologies)转染到孤雄单倍体胚胎干细胞中。转染48小时之后，通过流式(FACS AriaII,BD Biosciences)将带有红色荧光蛋白的单倍体细胞分选出来后，以较低的密度铺下。生长4-5天后，挑取单克隆进行后续的建系。最后通过PCR目的基因进行测序的方法获得相应基因突变的细胞系。
如涉及基因的敲入，则需构建双链的DNA供体。
双链DNA供体制备：
扩增到编码EGFP、mCherry或ECFP的序列，然后相应地连接到pMD19-T载体上得到pMD19-T-EGFP/mCherry/ECFP。随后，将目的基因的左右同源臂相应地插入到pMD19-T-EGFP/mCherry/ECFP的载体上。
1.5病毒的制备和双敲孤雄单倍体胚胎干细胞(DKO-AG-haESCs)的病毒感染
小鼠的sgRNA病毒文库和Cas9的病毒质粒已有报道(Cong,L.,Ran,F.A.,Cox,D.,Lin,S.,Barretto,R.,Habib,N.,Hsu,P.D.,Wu,X.,Jiang,W.,Marraffini,L.A.,et al.(2013).Multiplex genome engineering using CRISPR/Cas systems.Science 339,819-823；Koike-Yusa,H.,Li,Y.,Tan,E.P.,Velasco-Herrera Mdel,C.,and Yusa,K.(2014).Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.Nature biotechnology 32,267-273, 2014)，本发明所用小鼠sgRNA病毒文库和Cas9的病毒质粒由Addgene公司提供。为了制备病毒，HEK293T提前传代到10cm培养皿中，用
Figure PCTCN2015083165-appb-000033
2000Reagent(Invitrogen，Life Technologies)转染3ug的病毒质粒(sgRNA慢病毒文库或Cas9慢病毒)和9μg of ViraPower Lentiviral Packaging Mix慢病毒包装混合物(Invitrogen)转染到HEK293T细胞中。转染72小时后收集上清，用慢病毒浓缩液Lenti-Concentin virus precipitation solution(SBI)进行浓缩，随后就可以保存到-80℃。
Cas9慢病毒感染：用8ug/ml polybrene(Sigma)和包装好的Cas9慢病毒感染含108个DKO-AG-haESCs的细胞悬液48小时，然后用10ug/ml的blasticidin(Sigma)进行加药筛选3天，剩余的抗药克隆即为Cas9慢病毒整合的细胞系。
sgRNA慢病毒文库感染：用8ug/ml polybrene(Sigma)和包装好的CRISPR-sgRNA文库慢病毒感染含108个DKO-AG-haESCs的细胞悬液，48小时之后换成1ug/ml puromycin(Invitrogen))的培养基进行药物筛选，筛选2天后得到的阳性即为携带sgRNA文库慢病毒整合的细胞系。
Cas9慢病毒与sgRNA文库慢病毒双感染：
先制备Cas9慢病毒整合的细胞系，而后进一步感染sgRNA慢病毒文库，以1ug/ml puromycin(Invitrogen))的培养基进行药物筛选，筛选2天后得到的阳性即为Cas9慢病毒和sgRNA文库慢病毒整合的细胞系。
如果DKO-AG-haESCs细胞系只感染了sgRNA慢病毒文库，那么细胞就没有Cas9的表达，这个时候我们需要再细胞水平上进行pX330-mCherry质粒的转染，以实现基因组的编辑。1.6亚硫酸盐甲基化测序
1)将小鼠的DNA与15ul 2％LMP agarose(低熔点琼脂糖)包成beads，加入460ul DNA digestion buffer，再加入40ul proteinase K到每个样品，50℃过夜孵育消化样品。
2)TE洗3遍后，用bisulfite溶液与beads反应，50℃孵育4h-8h。
3)以beads为模板进行巢式PCR，PCR产物进行回收后与PMD19-T载体进行连接，转化，涂板
4)每个样品挑取10个菌落进行测序。
若采用EZ DNA methylation Gold kit(ZYMO Research)试剂盒，只需要准备好相应量的DNA，然后后续的所有过程根据试剂盒的步骤进行操作即可。得到的试剂盒回收产物，以此为模板进行PCR，产物回收，与PMD19-T载体进行连接，转化，涂板。选取平板的10个菌液进行测序。
1.7荧光定量PCR
细胞或器官的总量RNA用Trizol reagent(Invitrogen)抽提，然后用1ug的总RNA用First Strand cDNA Synthesis kit(TOYOBO)进行反转录成cDNA。实时荧光定量PCR反应用SYBR Green Realtime PCR Master Mix(TOYOBO)，并在Bio-Rad CFX96仪器上完成，每组样品设立3个重复。所有的基因表达水平均以管家基因Gapdh的表达水平为内参。
1.8Cobra Assay
1)取100ng的样品DNA，与TaqI快速限制性内切酶(fementas公司)(T/CGA)进行酶切反应15min。
2)进行琼脂糖凝胶电泳，即可。
1.9ICAHCI、ROSI和胚胎移植构建半克隆小鼠
胞浆内孤雄单倍体胚胎干细胞注射(intracytoplasmic AG-haESCs injection,ICAHCI)：
为获得半克隆(SC)胚胎，将AG-haESCs用含有0.05μg/ml秋水仙素的培养基处理8h以将细胞同步到M期，然后进行卵胞浆注射。消化好的AG-haESCs用HEPES-CZB培养液洗3遍，然后用3％(w/v)聚乙烯吡咯烷酮(polyvinylpyrrolidone，PVP)的HEPES-CZB培养液重悬。用Piezo显微操作仪将每个M期的AG-haESCs的细胞核注入MII卵子中。重构好的胚胎先在CZB培养液中培养1h，然后用不含CB的激活液激活5-6h。激活之后，所有的重构胚胎在KSOM培养液于37℃，5％CO2条件下进行培养。ICAHCI胚胎在KSOM培养液中培养24h就能到达2细胞期胚胎。
ROSI(球形精子注射)：
按照已报道的方法(Kishigami,S.,Wakayama,S.,Nguyen,V.T.,and Wakayama,T.(2004).Similar time restriction for intracytoplasmic sperm injection and round spermatid injection into activated oocytes for efficient offspring production.Biology of reproduction 70,1863-1869)进行操作。
由ICAHCI或ROSI获得的每15-20个2细胞胚胎)移入到0.5dpc(交配后0.5天)假孕ICR小鼠的每个子宫中。母鼠在怀孕19.5天后进行剖腹产或自然生产。剖腹产是针对WT AG-haESCs或单个DMR敲除的AG-haESCs所获得的重构胚胎，到期的胎儿快速从母鼠的子宫中剥离出来。针对ROSI或DKO-AG-haECs所得到的胚胎，怀孕19.5天的母鼠可以进行自然生产。清除掉出生小鼠身上的液体后，小鼠放在有氧气的温箱中，而存活的小鼠则后续由代孕母鼠进行养育。
1.10RNA-seq和基因表达分析
总RNA的RNA-seq建库是根据illumina官方的TreSeq RNA Sample Prep v2Guide进行的。完成后在中科院-马普学会计算生物学研究所计算组学中心的Illumina HiSeq 2000仪器上进行深度测序。2个WT AG-haESCs、
Figure PCTCN2015083165-appb-000034
Figure PCTCN2015083165-appb-000035
以及球形精子共6个样本进行后续的分析
基因表达量使用的RPKM，具体的算法是和(Yang,L.,Duff,M.O.,Graveley,B.R.,Carmichael,G.G.,and Chen,L.L.(2011).Genomewide characterization of non-polyadenylated RNAs.Genome biology 12,R16)一致。
计算差异表达基因的p-value时使用的是(Yang et al.,Genome boil 2011)中waldscore计算的方法，选择abs(waldscore)>1.96(即p-value<0.05)，然后筛选差异表达基因。
1.11RRBS(简化表观亚硫酸氢盐测序)
RRBS的建库是R是根据Illumina官方的标准protocol制备的。然后用Illumina HiSeq 2000进行测序(Gu,H.,Smith,Z.D.,Bock,C.,Boyle,P.,Gnirke,A.,and Meissner,A.(2011).Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling.Nature protocols 6,468-481.)。所有的测序读值与小鼠的基因组进行比对分析。
1.12基因型鉴定方法
抽提好的基因组DNA用相应的引物进行PCR，然后PCR产物进一步进行琼脂糖凝胶电泳分析。
实施例1
基于H19 DMR单敲除的孤雄单倍体胚胎干细胞系构建半克隆小鼠
A.构建孤雄单倍体胚胎干细胞系：
精子来源：H19 Δ3.8kb KO小鼠(C57/B6背景,纯合)；
卵子来源：B6D2F1(C57BL/6X DBA2)雌性小鼠
采用前述1.3的方法，将H19 Δ3.8kb小鼠的单倍体精子头注入去核的卵细胞(图1A)，获得重构的囊胚，构建孤雄单倍体胚胎干细胞系。从250颗重构的囊胚中建立了3株单倍体细胞系(命名为
Figure PCTCN2015083165-appb-000036
)(图1B-1D)。
B.构建半克隆小鼠：
采用前述1.9的方法，用
Figure PCTCN2015083165-appb-000037
细胞作为ICAHCI的供体，利用这3株
Figure PCTCN2015083165-appb-000038
细胞系将重构的1443颗2-细胞胚胎移植到假孕母鼠中。最后，通过将怀孕19.5天的母鼠进行剖腹产，得到了86只健康存活的半克隆小鼠和39只生长阻滞的小鼠(图1E，表1)。正常半克隆小鼠的出生效率大约为5.9％，然而，
Figure PCTCN2015083165-appb-000039
重构的胚胎依旧有约2.7％的半克隆小鼠发育不正常。
以野生型AG-haESCs作为ICAHCI的供体构建半克隆小鼠。正常半克隆小鼠的出生效率大约为0.7-1.8％。
C.Gtl2表达检测
采用前述1.7的方法通过荧光定量PCR检测
Figure PCTCN2015083165-appb-000040
来源的生长阻滞型小鼠主要器官中是否也出现Gtl2的异常高表达。
荧光定量用PCR引物序列：
Gtl2-F：TTGCACATTTCCTGTGGGAC
Gtl2-R：AAGCACCATGAGCCACTAGG
试验结果表明，相比于正常小鼠，生长阻滞型半克隆小鼠在大部分检测的器官中，均出现了Gtl2的高表达(图S1E)。
D.甲基化分析
实验方法：采用前述1.6和1.8的方法分析
所用引物：
IG DMR-BS-OF：
TTAAGGTATTTTTTATTGATAAAATAATGTAGTTT
IG DMR-BS-OR：
CCTACTCTATAATACCCTATATAATTATACCATAA
IG DMR-BS-IF：
TTAGGAGTTAAGGAAAAGAAAGAAATAGTATAGT
IG DMR-BS-IR：
TATACACAAAAATATATCTATATAACACCATACAA
甲基化分析的结果显示生长阻滞型的半克隆小鼠在Dlk1-Gtl2印记簇的甲基化差异位点IG-DMR区域出现明显的低甲基化(图S1F-G)。有趣的是，
Figure PCTCN2015083165-appb-000041
后期代数(
Figure PCTCN2015083165-appb-000042
p16)相比于它的早期代数(
Figure PCTCN2015083165-appb-000043
p7)出现了IG-DMR甲基化印记更加严重的丢失，最后产生生长阻滞型小鼠的比率也明显升高(图S1H，表S1)。说明Gtl2的异常表达可能是导致
Figure PCTCN2015083165-appb-000044
细胞获得的半克隆小鼠发育失败的另外一个重要因素。
实施例2
基于H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建半克隆小鼠
A.H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建：
采用前述1.4的方法构建。
在Dlk1和Gtl2之间设计了敲除4.15kb IG-DMR的2个sgRNA(命名为IG-DMR-sgRNA1和IG-DMR-sgRNA2)(图1F)。
IG-DMR-sgRNA1序列：CGTACAGAGCTCCATGGCAC(SEQ ID NO:1)
IG-DMR-sgRNA2序列：CTGCTTAGAGGTACTACGCT(SEQ ID NO:2)
构建表达Cas9和IG-DMR-sgRNAs的质粒pX330-mCherry，转染到
Figure PCTCN2015083165-appb-000045
细胞中，最终获得了71个孤雄单倍体胚胎干细胞系。目的基因PCR产物测序(IG-DMR deletion check–F:TGTGCAGCAGCAAAGCTAAG IG-DMR deletion check–R:ATACGATACGGCAACCAACG)发现有58个细胞系的IG-DMR得到了成功的敲除(命名
Figure PCTCN2015083165-appb-000046
到
Figure PCTCN2015083165-appb-000047
)(图1G，S2A和S2B)。对
Figure PCTCN2015083165-appb-000048
进行脱靶分析显示总共22个潜在的脱靶位点均没有发生突变(表S2)，这些位点的预测是根据之前报道的软件对整个小鼠基因组进行搜索(Hsu,P.D.,Scott,D.A.,Weinstein,J.A.,Ran,F.A.,Konermann,S.,Agarwala,V.,Li,Y.,Fine,E.J.,Wu,X.,Shalem,O.,et al.(2013).DNA targeting specificity of RNA-guided Cas9nucleases.Nature biotechnology31,827-832)。
B.构建半克隆小鼠：
采用前述1.9的方法，用
Figure PCTCN2015083165-appb-000049
细胞作为ICAHCI的供体构建半克隆小鼠，结果显示有22.3％的半克隆胚胎都能发育到期(图1H，图S2C，表1，表S1)，与我们实验室(表1)或其他实验室报道(Kishigami,S.,Wakayama,S.,Nguyen,V.T.,and Wakayama,T.(2004).Similar time restriction for intracytoplasmic sperm injection and round spermatid injection into activated oocytes for efficient offspring production.Biology of reproduction 70,1863-1869.)的ROSI出生率相似。更者，相比于野生型AG-haESCs和
Figure PCTCN2015083165-appb-000050
通过ICAHCI所获得的半克隆小鼠需要进行剖腹产，而
Figure PCTCN2015083165-appb-000051
重构的胚胎移植到假孕母鼠后，假孕母鼠能够进行自然生产，而且出生的半克隆小鼠均健康地活着。这些数据说明
Figure PCTCN2015083165-appb-000052
细胞通过敲除IG-DMR后成功建立起类似球形精子的特性，并且高效地产生半克隆小鼠。
实施例3
基于IG-DMR单敲除的孤雄单倍体胚胎干细胞系构建半克隆小鼠
A.构建孤雄单倍体胚胎干细胞系：
精子来源：IG-DMR KO小鼠；
卵子来源：B6D2F1(C57BL/6X DBA2)雌性小鼠
采用前述1.3的方法，将IG-DMR KO小鼠的单倍体精子头注入去核的卵细胞，获得重构的囊胚，构建孤雄单倍体胚胎干细胞系。在总共建立的8株单倍体细胞系中，有2株细胞系是携带IG-DMR敲除(命名为
Figure PCTCN2015083165-appb-000053
和
Figure PCTCN2015083165-appb-000054
)
B.构建半克隆小鼠：
采用前述1.9的方法，用
Figure PCTCN2015083165-appb-000055
细胞作为ICAHCI的供体构建半克隆小鼠，发现
Figure PCTCN2015083165-appb-000056
细胞并不是产生半克隆小鼠的有效供体(表1、表S1)，并且很难得到健康正常的SC小鼠(499个移植的胚胎只获得了4只正常的SC小鼠)(图2A、S2E)。更者，大部分的小鼠都属于生长阻滞型。
C.甲基化分析
实验方法：采用前述1.6和1.8的方法分析
所用引物：
H19 DMR-BS-OF:5'GAGTATTTAGGAGGTATAAGAATT 3'
H19 DMR-BS-OR:5'ATCAAAAACTAACATAAACCCCT 3'
H19 DMR-BS-IF:5'GTAAGGAGATTATGTTTATTTTTGG 3'
H19 DMR-BS-IR:5'CCTCATTAATCCCATAACTAT 3'
结果显示，
Figure PCTCN2015083165-appb-000057
细胞系的H19 DMR甲基化出现擦除，并且在生长阻滞的小鼠中完全丢失其甲基化(图2B、S2F和S2G)。
实施例4
基于H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建半克隆小鼠
A.H19 DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建：
采用前述1.4的方法构建。
敲除H19的Δ3.8kb DMR的sgRNA序列：
H19-3.8K sgRNA-1:CATGAACTCAGAAGAGACTG(SEQ ID NO:3)
H19-3.8K sgRNA-2:AGGTGAGAACCACTGCTGAG(SEQ ID NO:4)
通过CRISPR-Cas9的方法在前述实施例来源的
Figure PCTCN2015083165-appb-000058
细胞系中敲除H19的Δ3.8kb DMR(参考Thorvaldsen,J.L.,Mann,M.R.,Nwoko,O.,Duran,K.L.,and Bartolomei,M.S.(2002).Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting.Molecular and cellular biology 22,2450-2462)，并成功获得了13株DKO-AG-haESC细胞系(命名为
Figure PCTCN2015083165-appb-000059
到
Figure PCTCN2015083165-appb-000060
)(图2C、S2H和表S2)。
B.构建半克隆小鼠：
采用前述1.9的方法，用前述其中2株细胞作为ICAHCI的供体构建半克隆小鼠，结果 显示与
Figure PCTCN2015083165-appb-000061
细胞获得健康SC小鼠的能力极为相似(图S2I、2J和表1、表S1)。
实施例5
基于H19-DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建半克隆小鼠
A.H19-DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系构建：
初始细胞：WT-AG-haESCs细胞系AGH-OG-3的第21代AGH-OG-3细胞
已有报道表明，该细胞系第22代已经基本上丧失获得健康的半克隆小鼠的能力(Yang,H.,Shi,L.,Wang,B.A.,Liang,D.,Zhong,C.,Liu,W.,Nie,Y.,Liu,J.,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617)
将sgRNA的oligo进行退火，然后将H19和IG-DMR的sgRNA分别与BbsI酶切的px330-mCherry质粒进行连接转化。将测序正确的菌液进行质粒抽提，以备后续转染。
将第21代的AGH-OG-3细胞系中转入上述获得的质粒：
最终获得了12株同时敲出了H19和Gtl2DMR的孤雄单倍体胚胎干细胞系(命名
Figure PCTCN2015083165-appb-000062
到
Figure PCTCN2015083165-appb-000063
)(图2D、S3A和表S2)。
B.构建半克隆小鼠：
采用前述1.9的方法，用前述
Figure PCTCN2015083165-appb-000064
细胞作为ICAHCI的供体构建半克隆小鼠。让人兴奋的是，通过将其中的2株细胞系注入到MII卵子中，大约有17％的半克隆胚胎能够发育到期(图S3B、S3C、表1和表S1)。这说明之前已经完全丧失获得SC小鼠能力的WT-AG-haESCs在敲除H19-DMR和IG-DMR之后能够重新获得其球形精子类似的特性。
实施例6
3种类型的DKO-AG-haESCs获得的SC小鼠基因型确定
基因型鉴定方法：采用前述1.12的方法
H19-DMR deletion check–F2：GTGGTTAGTTCTATATGGGG
H19-DMR deletion check–R2：TCTTACAGTCTGGTCTTGGT
IG-DMR deletion check–F:TGTGCAGCAGCAAAGCTAAG
IG-DMR deletion check–R:ATACGATACGGCAACCAACG
采用前述实施例2、4、5的三种方法，构建的三种类型H19-DMR及IG-DMR双敲除的孤雄单倍体胚胎干细胞系，总获得了402只SC小鼠，移植的胚胎达到20.2％的小鼠出生效率。这些DKO-AG-haESCs来源的SC小鼠能够长大成年，并且能够产生后代。对7窝里33只新生小鼠后代进行基因型鉴定发现：13只携带有H19-DMR敲除，11只为WT(图2E)。而另外的9只小鼠有6只为IG-DMR敲除，3只为H19和Gtl2DMR双敲除。这9只小鼠出生不久便死去，这和之前报道的IG-DMR母系遗传会出现出生前或出生后致死的表型相一致(Lin,S.P.,Youngson,N.,Takada,S.,Seitz,H.,Reik,W.,Paulsen,M.,Cavaille,J.,and Ferguson-Smith,A.C.(2003).Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2imprinted cluster on mouse chromosome 12.Nature genetics 35,97-102)。
实施例7
AG-haESCs的基因表达检测及甲基化分析
采用前述1.10和1.7的方法分别进行RNA-seq及基因表达分析。
试剂：SYBR-Green(TOYOBO)
Q-PCR所用引物：
Gapdh-F：CACTCTTCCACCTTCGATGC
Gapdh-R：CTCTTGCTCAGTGTCCTTGC
Igf2-F：CTAAGACTTGGATCCCAGAACC
Igf2-R：GTTCTTCTCCTTGGGTTCTTTC
Gtl2-F：TTGCACATTTCCTGTGGGAC
Gtl2-R：AAGCACCATGAGCCACTAGG
Dlk-F:ACTTGCGTGGACCTGGAGAA
Dlk-R:CTGTTGGTTGCGGCTACGAT
H19-F:CATGTCTGGGCCTTTGAA
H19-R:TTGGCTCCAGGATGATGT
采用前述1.11的方法进行全基因组甲基化水平分析
Q-PCR的结果显示在DKO-AG-haESCs中H19和Gtl2的表达发生了下调，而Igf2和Dlk1的表达则上调(图S3D)。比较了DKO-AG-haESCs和正常的AG-haESCs以及球形精子的基因表达谱。基于RNA-seq的数据进行聚类发现DKO-AG-haESCs与WT-AG-haESCs表达谱有极高的相似性，但与球形精子相距甚远(图2F、S3E)。进一步分析DKO-AG-haESCs和WT-AG-haESCs的印记基因表达谱，发现DKO-AG-haESCs和WT-AG-haESCs所有的印记基因都有极其相似的表达水平(图2G)。为了进一步评估其表观遗传，进行了简化的表观亚硫酸氢盐测序(RRBS)从全基因组水平范围内看其甲基化水平。正如图2H、S3F和S3G所显示，H19和Gtl2DMRs的敲除并没有改变所有检测到基因和印记基因启动子区域的甲基化水平。总之，我们的结果显示H19和Gtl2DMRs是AG-haESCs获得球形精子类似特性的两大主要障碍。
实施例8
携带多基因修饰的DKO-AG-haESCs产生半克隆小鼠
A.携带多基因修饰的DKO-AG-haESCs构建：
采用前述1.4的方法构建。
初始细胞：DKO-AG-haESCsTET家族基因敲除采用实施例4制备的DKO-AG-haESCs，p53家族基因敲除采用实施例2制备的DKO-AG-haESCs)
突变目标：Tet1、Tet2、Tet3、p53家族突变
构建过程：
Tet-TKO-DAH的构建过程：
将Tet1、Tet2、Tet3的sgRNA分别退火，然后分别连接到BbsI酶切的px330-mCherry质 粒上，通过测序挑出连入sgRNA的阳性质粒。
Tet1 sgRNA序列：GGCTGCTGTCAGGGAGCTCA(SEQ ID NO:5)
Tet2 sgRNA序列：GAAAGTGCCAACAGATATCC(SEQ ID NO:6)
Tet3 sgRNA序列：AAGGAGGGGAAGAGTTCTCG(SEQ ID NO:7)
将表达Tet1、Tet2、Tet3的sgRNA的质粒共同转入DKO-AG-haESCs细胞系中，分选出mCherry阳性的细胞，铺到培养皿中。生长5天后，挑取克隆，传代扩增。建立好的细胞系通过PCR产物测序鉴定Tet1、Tet2、Tet3的突变情况。
测序引物：
Tet1check-F：GCCCCTGTTGTCTTATACGTT
Tet1check-R：CATTCGCCTCAGGACCAC
Tet2check-F：CCGCCACAAGAAAATATGTCC
Tet2check-R：AGCTAACTCTGGCAAACACC
Tet3check-F：CAGAGTGGCCTCAGTTTCCC
Tet3check-R：ACAACTTTTACCCAGGAGTCACAC
p53-TKO-DAH的构建过程：
将p53、p63、p73的sgRNA分别退火，然后分别连到BbsI酶切的px330-mCherry质粒上，通过测序挑出连入sgRNA的阳性质粒。
p53 sgRNA序列:CACCTGGGCTTCCTGCAGTC(SEQ ID NO:8)
p63 sgRNA序列:TGGGCCCGGGTAATCTGTGT(SEQ ID NO:9)
p73 sgRNA序列:TGTCGATAGGAGTCAACCAA(SEQ ID NO:10)
将表达p53、p63、p73的sgRNA的质粒共同转入DKO-AG-haESCs细胞系中，分选出mCherry阳性的细胞，铺到培养皿中。生长5天后，挑取克隆，传代扩增。建立好的细胞系通过PCR产物测序鉴定p53、p63、p73的突变情况。
P53 check-F:CCCCTGTCATCTTTTGTCCCT
P53 check-R:AAGAGAGTTCCACGTCCCCTG
P63 check-F:CACACCAAATAATGCCAATT
P63 check-R:CAGACTCTCTTACCGTCCAG
P73 check-F:GACCCACTTCTAAACCTGCC
P73 check-R:CCATACCTCCTGTGCTCCTG
B.构建半克隆小鼠：
采用前述1.9的方法，用前述构建的细胞作为ICAHCI的供体构建半克隆小鼠。
C.结果
本实例用CRISPR-Cas9的方法在DKO-AG-haESCs突变Tet1、Tet2和Tet3。将构建好表达Cas9以及Tet1、2和3 3个sgRNAs(图3A)(参考Wang,H.,Yang,H.,Shivalila,C.S.,Dawlaty,M.M.,Cheng,A.W.,Zhang,F.,and Jaenisch,R.(2013).One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell 153,910-918)的质粒转入DKO-AG-haESCs中，最终建立了56株DKO-AG-haESCs，Tet1、2和3基因的PCR 产物测序发现有18株成功地发生了三基因的突变(命名为Tet-TKO-DAH-1到Tet-TKO-DAH-18)(图3B-D和S4A)。其中4株细胞系的ICAHCI结果表明能够以相应的效率获得Tet1、2和3突变的SC小鼠(图3E、S4B、表1和表S3)。p53家族突变的DKO-AG-haESCs通过ICAHCI也能有效地获得相应的SC小鼠(图S4D、S4E、表1和表S3)。这些结果显示：WT-AG-haESCs在体外进行基因操作后无法获得存活的SC小鼠，但是DKO-AG-haESCs在基因编辑操作之后仍能够有效稳定地产生半克隆小鼠。
实施例9
DKO-AG-haESCs进行基因编辑并产生半克隆小鼠
A.DKO-AG-haESCs基因编辑细胞系的构建
初始细胞：DKO-AG-haESCs(是实施例4制备)
构建过程：
Tet1&3-KI-DAH的构建过程：
将Tet1和Tet3的sgRNA分别退火，然后连到BbsI酶切的px330-mCherry质粒上，通过测序挑出连入sgRNA的阳性质粒。针对Tet1-EGFP和Tet3-ECFP供体的制备，分别以pEGFP-N1质粒和pECFP-N1质粒为模板，(引物为：P2A-fluorescence F：GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCGAGGAG
P2A-fluorescence R：CTTGTACAGCTCGTCCATG)扩增到编码EGFP、或ECFP的序列，然后相应地连接到pMD19-T载体的多克隆位点上，获得pMD19-T-EGFP/ECFP的载体。随后，将目的基因的左右同源臂相应地插入到pMD19-T-EGFP/ECFP的载体中EGFP或ECFP基因的两侧。
同源臂序列如下：
TET1 LA(左同源臂)：(SEQ ID NO:11)
TET1 RA(右同源臂)：(SEQ ID NO:12)
TET3 LA(左同源臂)：(SEQ ID NO:13)
TET3 RA(右同源臂)：(SEQ ID NO:14)
将Tet1和Tet3的sgRNA质粒与Tet1-EGFP和Tet3-ECFP供体这4个质粒共同转入DKO-AG-haESCs细胞系中，分选出mCherry阳性的细胞，铺到培养皿中。生长5天后，挑取克隆，传代扩增。建立好的细胞系通过PCR的方法鉴定Tet1-EGFP和Tet3-ECFP的敲入情况。双敲入的细胞命名为Tet1&3-KI-DAH。
以下为PCR鉴定引物：
Tet1 LA-F：TTTGTGTCTATGAACTACCAGTGAG
Tet1 LA-F：CAGGCCCGGGGTTTTCTTC
Tet1 RA-F：CAACGAGAAGCGCGATCACA
Tet1 RA-F：TTTTGACTGATCCCAATTTGCCT
Tet3 LA-F：TGTTCACTGGTGAAGGCCAG
Tet3 LA-F：GAACAGCTCCTCGCCCTTG
Tet3 RA-F：TGAGCAAAGACCCCAACGAG
Tet3 RA-R:ATCGACAAACTTTGGGGCGA
Tet-TKI-DAH的构建过程：
将Tet2的sgRNA退火，然后连到BbsI酶切的px330-mCherry质粒上，通过测序挑出连入sgRNA的阳性质粒。
针对Tet2-mCherry供体的制备，以pmCherry-N1为模板，P2A-fluorescence F：GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCGAGGAG；P2A-fluorescence R：CTTGTACAGCTCGTCCATG为引物扩增到编码mCherry的序列，然后连接到pMD19-T载体上。随后，将目的基因的左右同源臂相应地插入到pMD 19-T-mCherry的载体上mCherry序列的两侧。
TET2 LA(左同源臂)：(SEQ ID NO:15)
TET2 RA(右同源臂)：(SEQ ID NO:16)
将Tet2的sgRNA质粒与Tet2-mCherry供体这2个质粒共同转入Tet1&3-KI-DAH细胞系中，分选出mCherry阳性的细胞，铺到培养皿中。生长5天后，挑取克隆，传代扩增。建立好的细胞系通过PCR的方法鉴定Tet2-mCherry的敲入情况。阳性的细胞克隆即为Tet-TKI-DAH细胞系。
鉴定引物：
Tet2 LA-F：CACACCCTTCACCAACAGACG
Tet2 LA-R：ATCTCGAACTCGTGGCCGTT
Tet2 RA-F：AAGACCACCTACAAGGCCAAG
Tet2 RA-R：GGTAGGCAAAGTGCTTTTCTAAGAC
B.构建半克隆小鼠：
采用前述1.9的方法，用前述构建的细胞作为ICAHCI的供体构建半克隆小鼠。
C.结果
本实例获得内源Tet1、Tet12和Tet3敲入不同荧光报告基团的DKO-AG-haESCs。首先，向DKO-AG-haESCs转入同时表达Cas9和Tet1、Tet3sgRNA(图3A)的质粒以及分别在Tet1和Tet3最后一个终止子融合了EGFP和ECFP报告基团的双链DNA供体载体(图3H和S5A)。总共150个DKO-AG-haESCs细胞系中，分别获得了10株Tet1-EGFP敲入和7株Tet3-ECFP敲入的细胞系(图S5B和S5C)。其中有一株细胞系同时携带Tet1-EGFP和Tet3-ECFP敲入，命名为Tet1&3-KI-DAH-1(图S5D和S5E)。ICAHCI结果显示携带Tet1-EGFP和Tet3-ECFP敲入的DKO-AG-haESCs与WT DKO-AG-haESCs有着相似的半克隆小鼠出生能力(图S5F、S5G、表1和表S3)。接下来，往Tet1&3-KI-DAH-1细胞系中转入表达Cas9和Tet2sgRNA的质粒以及在Tet2基因最后一个终止子融合了mCherry报告基团的双链DNA供体载体(图3A和3H)。从建立的130株细胞系，我们鉴定出了8株Tet2-RFP敲入的单倍体细胞系(命名Tet-TKI-DAH-1to Tet-TKI-DAH-8)(图3I和S5H)。最后，通过ICAHCI的方法验证Tet-TKI-DAH细胞系的发育潜能。相一致地，Tet-TKI-DAH能够通过注射到卵子里高效地获 得存活的半克隆小鼠(图S5S、S5J、表1和表S3)。总之，这些结果表明：利用DKO-AG-haESCs进行多基因遗传操作然后通过ICAHCI获得相应遗传学特性的SC小鼠是一种可行的途径。
实施例10
携带sgRNA文库的DKO-AG-haESCs大规模获得杂合突变的SC小鼠
A.携带sgRNA文库的DKO-AG-haESCs的构建
初始细胞：
Figure PCTCN2015083165-appb-000065
采用前述1.5的方法进行病毒制备及用全基因组sgRNA慢病毒文库感染DKO-AG-haESCs后，转染表达Cas9的pX330-mCherry质粒，最终构建获得携带sgRNA文库的DKO-AG-haESCs。
B.构建半克隆小鼠：
采用前述1.9的方法，用前述构建的细胞作为ICAHCI的供体构建半克隆小鼠。
半克隆小鼠携带sgRNA情况检测：
sgRNA的检测是通过特定引物PCR扩增，然后琼脂糖凝胶电泳跑胶看是否有条带。
Lenti-sg-F：GTTACTCGAGCCAAGGTCGG
Lenti-sg-R：GACTCGGTGCCACTTTTTCA
等位基因突变检测：
根据sgRNA测序鉴定出插入相应的基因，然后在目的基因sgRNA附近常规方法设计上下游引物，PCR，然后测序。
C.结果
目前，全基因组水平的sgRNA文库已经成功建立，并且运用于人和小鼠细胞水平的基因功能缺失筛选中(Koike-Yusa,H.,Li,Y.,Tan,E.P.,Velasco-Herrera Mdel,C.,and Yusa,K.(2014).Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.Nature biotechnology 32,267-273)。本试验证明，DKO-AG-haESCs能够携带sgRNA文库，能通过ICAHCI简单地一步法获得大量的突变小鼠模型(图4A)(这一方法命名为Lenti-sgRNA+pX330)。本试验使用刚建立好并且比较清楚的小鼠慢病毒文库。这个文库针对19,150小鼠编码蛋白的基因设计了87,897个sgRNA。1.0×107FACS富集的单倍体细胞系
Figure PCTCN2015083165-appb-000066
用全基因组sgRNA慢病毒文库进行感染。2天之后，这些感染的细胞用puromycin处理7天，随后转染表达Cas9的pX330-mCherry质粒。表达mCherry的单倍体细胞指示着Cas9的成功转染表达，这种单倍体进行FACS富集后用于进行ICAHCI实验(图4B)。为了检测单倍体的基因是否被CRISPR-Cas9诱导突变，随机挑取了7个单倍体细胞克隆。所有检测的细胞克隆携带有一个sgRNA，DNA测序也发现这些目的基因被突变，这说明单倍体细胞能成功地用CRISPR-Cas9诱导的基因突变。
通过3次独立ICAHCI实验，获得了114只半克隆小鼠(图4E和表2)，其中有82只小鼠携带有一个sgRNA(图4F)。sgRNA的PCR产物测序发现43只SC小鼠携带有目的基因一个等位基因的突变，其中有39个是携带导致移码的插入或敲除突变(indel)进而造成一个等位基因的功能缺失(图4G)。有趣地，所有的突变都是一种基因型，说明单倍体细胞在Cas9的瞬时表达后实现了基因的突变。而剩余的39只，虽然携带一个sgRNA，但是在需要突变 的位点没有出现DNA切割，这个效率和之前在人细胞(Zhou,Y.,Zhu,S.,Cai,C.,Yuan,P.,Li,C.,Huang,Y.,and Wei,W.(2014).High-throughput screening of a CRISPR/Cas9library for functional genomics in human cells.Nature 509,487-491)或小鼠胚胎(Wu,Y.,Liang,D.,Wang,Y.,Bai,M.,Tang,W.,Bao,S.,Yan,Z.,Li,D.,and Li,J.(2013).Correction of a genetic disease in mouse via use of CRISPR-Cas9.Cell Stem Cell 13,659-662)中所观察到的相一致。这些数据说明携带sgRNA文库的DKO-AG-haESCs瞬时转染表达Cas9通过ICAHCI可以实现遗传突变向SC小鼠的引入，最终一步获得大量杂合突变的小鼠。
实施例11
DKO-AG-haESCs一步法获得sgRNA介导的双等位基因突变小鼠
A.携带sgRNA的单倍体细胞的构建
初始细胞：
Figure PCTCN2015083165-appb-000067
用携带CRISPR-sgRNA的慢病毒文库采用1.5的方法感染DKO-AG-haESCs得到携带sgRNA文库慢病毒整合的细胞系。
B.构建半克隆小鼠：
通过ICAHCI将携带sgRNA的单倍体的细胞核注入成熟的卵子，然后再向重构的卵子里胞浆注射注入Cas9mRNA(这种方案命名为：Lenti-sgRNA+Cas9injection)。而后胚胎移植构建半克隆小鼠。
半克隆小鼠sgRNA检测：参考实施例9的方法。
等位基因突变检测：参考实施例9的方法。此外，还将目的基因的PCR产物连入pMD19-T载体中，然后挑克隆测序。
C.结果
本实验通过ICAHCI将携带sgRNA的单倍体注入成熟的卵子，然后再向重构的卵子里注入Cas9(这种方案命名为：Lenti-sgRNA+Cas9injection)。总共出生了45只携带一个sgRNA的SC小鼠，其中有22只发生了基因突变(表2)。目的基因的PCR产物测序发现有10只小鼠只有单个等位基因的修饰，而另外12只则是双等位基因突变(占所有出生SC小鼠的23.5％)。将7只双等位基因突变的SC小鼠进行TA克隆测序分析发现大约63％的克隆都是携带插入缺失突变(表S4)。
实施例12
DKO-AG-haESCs一步法获得sgRNA介导的双等位基因突变小鼠
A.采用实施例10A的方法构建携带sgRNA的单倍体细胞，再将pX330-mCherry质粒瞬转进携带sgRNA的单倍体细胞。
B.构建半克隆小鼠：
通过ICAHCI将A中获得单倍体细胞的细胞核注射到卵子之后接着再用胞浆注射的方法注入Cas9mRNA，而后胚胎移植构建半克隆小鼠。
半克隆小鼠sgRNA检测：参考实施例9的方法。
等位基因突变检测：参考实施例11的方法。
C.结果
本试验将pX330-mCherry质粒瞬转进携带sgRNA的单倍体细胞，注射到卵子之后接着再注入Cas9mRNA(图S6A和S6B)(这种方案命名为：Lenti-sgRNA+pX330+Cas9injection)。总共获得27只携带一个sgRNA的SC小鼠，22只携带基因突变(图S6C、S6D和表2)，其中的突变小鼠中有13只携带有双等位基因突变(图S6E)(占所有SC小鼠的41.9％)，另外9只小鼠为单等位基因突变。对其中的5只双等位基因突变小鼠进行TA克隆和测序结果显示约79％的克隆都是存在插入缺失突变(图S6F、S6G和表S4)
实施例13
DKO-AG-haESCs一步法获得sgRNA介导的双等位基因突变小鼠
A.Cas9和sgRNA文库持续表达的DKO-AG-haESCs的构建
初始细胞：
Figure PCTCN2015083165-appb-000068
采用Lenti-Cas9+lenti-sgRNA法构建细胞系的具体步骤：采用1.5中“Cas9慢病毒与sgRNA文库慢病毒双感染”的方法制备。
Cas9慢病毒和sgRNA文库慢病毒整合的细胞系进一步进行流式分选富集单倍体，以用于后续的ICAHCI操作。
B.构建半克隆小鼠：
采用前述1.9的方法，将Cas9和sgRNA文库持续表达的DKO-AG-haESCs作为ICAHCI的供体构建半克隆小鼠。
等位基因突变检测：参考实施例11的方法。
鉴定小鼠突变情况的引物举例：
Polm check-F：TCCGATGGGAAGCCAAAAGC
Polm check-R：CGTACCGCAACCGCGAAGTA
Scube1 check-F：CCATAATAATCCACTTCCAT
Scube1 check-R:CCAACCCCTGTCCACTACCT
C.结果
本试验通过两轮药物筛选获得Cas9和sgRNA文库持续表达的DKO-AG-haESCs，然后用ICAHCI技术产生SC小鼠(图5A-D)(这一方案命名：Lenti-Cas9+lenti-sgRNA)。ICAHCI重构的1453颗胚胎中有272颗(18.7％)发育到期(图5E)，基本上和WT DKO-AG-haESCs或携带不同基因修饰的DKO-AG-haESCs有相似的出生效率(表2)，这说明DKO-AG-haESCs经过多次基因遗传操作后仍不影响其获得SC小鼠的能力。总共有224只SC小鼠携带一个sgRNA，并用于后续的基因型分析(图5F和表2)。结果显示有143只SC小鼠发生基因突变，其中有83只是双等位基因突变小鼠(图5G)。获得了60只单等位基因突变的SC小鼠，虽然推测这些小鼠的Cas9应该是处于持续表达的，但由于病毒载体的沉默导致在SC小鼠发育的不同阶段出现Cas9的转录沉默。通过对26只双等位基因突变的SC小鼠进行TA克隆和测序显示约66.3％的克隆都是发生了插入缺失突变(图5H、I)。为了进一步确定SC小鼠整个身体的突变效率，分析了4只SC小鼠不同器官(包括大脑、心脏、肾脏、肝脏和肺)的基因突变情况，发现这些器官均为双等位基因突变。最后对一只携带Scube1基因突变SC小鼠 的不同器官进行TA克隆和测序。结果显示有约80％的克隆都发生插入缺失突变(图5J)。有趣地，这只SC小鼠在出生一小时内便死去，这和之前的关于Scube1突变小鼠出生后不久死去的研究报道相一致(Tu,C.F.,Yan,Y.T.,Wu,S.Y.,Djoko,B.,Tsai,M.T.,Cheng,C.J.,and Yang,R.B.(2008).Domain and functional analysis of a novel platelet-endothelial cell surface protein,SCUBE1.The Journal of biological chemistry 283,12478-12488)。总之，上述实验结果为利用携带sgRNA文库的DKO-AG-haESCs获得大量突变小鼠进而实现小鼠水平上基于基因敲除的大规模筛选这一可能提供了充分的证据，大大简化DKO-AG-haESCs介导的基于基因敲除的筛选过程。
表1、ICAHCI胚胎体内发育的情况汇总
Figure PCTCN2015083165-appb-000069
Figure PCTCN2015083165-appb-000070
a:AG-haESCs：本发明制备。见方法1.3
b:AG-haESCs：来源于现有技术(Yang,H.,Shi,L.,Wang,B.A.,Liang,D.,Zhong,C.,Liu,W.,Nie,Y.,Liu,J.,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617)是该文献发表者馈赠
c:
Figure PCTCN2015083165-appb-000071
Cells相比WT AG-haESCs p<0.05
d:
Figure PCTCN2015083165-appb-000072
Cells相比WT AG-haESCs p>0.05
e:DKO-AG-haESCs相比WT AG-haESCs p<0.001
表S1不同AG-haESCs ICAHCI胚胎的体内发育情况
Figure PCTCN2015083165-appb-000073
Figure PCTCN2015083165-appb-000074
a:AG-haESCs：参考现有技术自制.
b:AG-haESCs：来源于现有技术(Yang,H.,Shi,L.,Wang,B.A.,Liang,D.,Zhong,C.,Liu,W.,Nie,Y.,Liu,J.,Zhao,J.,Gao,X.,et al.(2012).Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.Cell 149,605-617)
表S3基因修饰的DKO-AG-haESCs ICAHCI胚胎的体内发育情况
Figure PCTCN2015083165-appb-000075
Figure PCTCN2015083165-appb-000076
表2携带sgRNA文库的DKO-AG-haESCs ICAHCI胚胎的体内发育情况汇总
Figure PCTCN2015083165-appb-000077
Figure PCTCN2015083165-appb-000078
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Figure PCTCN2015083165-appb-000079
Figure PCTCN2015083165-appb-000080
Figure PCTCN2015083165-appb-000081
Figure PCTCN2015083165-appb-000082
Figure PCTCN2015083165-appb-000083
Figure PCTCN2015083165-appb-000084

Claims (17)

  1. 一种孤雄单倍体胚胎干细胞，所述孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR被敲除。
  2. 如权利要求1所述孤雄单倍体胚胎干细胞，其特征在于，所述孤雄单倍体胚胎干细胞来源于哺乳动物，较优选来源于啮齿动物，优选来源于鼠，最优选来源于小鼠。
  3. 一种孤雄单倍体胚胎干细胞的制备方法，包括将孤雄单倍体胚胎干细胞的H19 DMR及IG-DMR敲除获得所述孤雄单倍体胚胎干细胞。
  4. 如权利要求3所述的孤雄单倍体胚胎干细胞的制备方法，其特征在于，采用CRISPR/Cas9介导的基因操作敲除H19 DMR及IG-DMR。
  5. 如权利要求3所述的孤雄单倍体胚胎干细胞的制备方法，其特征在于，还包括对所述孤雄单倍体胚胎干细胞进行了单个或者多个感兴趣目标基因的改造。
  6. 如权利要求1或2所述孤雄单倍体胚胎干细胞的用途，为用于构建基因改造的半克隆动物。
  7. 一种构建基因改造半克隆动物的方法，包括：将H19 DMR及IG-DMR被敲除的孤雄单倍体胚胎干细胞与卵细胞结合获得半克隆胚胎，培育所述半克隆胚胎获得半克隆动物。
  8. 如权利要求7所述构建基因改造半克隆动物的方法，其特征在于，以H19 DMR及IG-DMR被敲除的孤雄单倍体胚胎干细胞为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎。
  9. 如权利要求7所述构建基因改造半克隆动物的方法，其特征在于，所述H19 DMR及IG-DMR被敲除的孤雄单倍体胚胎干细胞的单个或者多个感兴趣目标基因经改造。
  10. 如权利要求7所述基因改造动物，其特征在于，所述半克隆动物为非人哺乳动物，较优选为啮齿动物，更优选为鼠，最优选为小鼠。
  11. 一种基因改造动物，为由权利要求7-10任一权利要求所述方法构建获得的半克隆动物，或者为权利要求7-10任一权利要求所述方法构建的半克隆动物的有性繁殖后代。
  12. 一种构建基因改造半克隆动物文库的方法，选自以下任一：
    方法一，包括下列步骤：
    1)以sgRNA慢病毒文库质粒制备的病毒颗粒感染权利要求1所述的孤雄单倍体 胚胎干细胞，获得携带sgRNA文库的孤雄单倍体胚胎干细胞文库；
    2)以携带sgRNA文库的孤雄单倍体胚胎干细胞库中的孤雄单倍体胚胎干细胞作为ICAHCI的供体，利用表达Cas9的载体和/或Cas9的mRNA，采用ICAHCI法获得半克隆胚胎文库；
    3)培育所述半克隆胚胎文库中的胚胎获得半克隆动物文库；
    方法二，包括下列步骤：
    1)以表达Cas9的慢病毒颗粒和sgRNA慢病毒文库制备的病毒颗粒感染权利要求1所述的孤雄单倍体胚胎干细胞，获得携带sgRNA文库以及Cas9持续表达的孤雄单倍体胚胎干细胞文库；
    2)以携带sgRNA文库以及Cas9持续表达的孤雄单倍体胚胎干细胞文库中的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎文库；
    3)培育所述半克隆胚胎文库中的胚胎获得半克隆动物文库。
  13. 如权利要求12所述构建基因改造半克隆动物文库的方法，其特征在于，所述方法一中的步骤2)可选自以下任一：
    方法A：
    向携带sgRNA文库的孤雄单倍体胚胎干细胞文库进一步转染表达Cas9的质粒，将获得的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法获得半克隆胚胎。
    方法B：
    以携带sgRNA文库的孤雄单倍体胚胎干细胞文库的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法注入成熟卵子，再向重构的卵子注入Cas9 mRNA获得半克隆胚胎。
    方法C：
    向携带sgRNA文库的孤雄单倍体胚胎干细胞文库进一步转染表达Cas9的质粒，将获得的孤雄单倍体胚胎干细胞作为ICAHCI的供体，采用ICAHCI法注入成熟卵子，再向重构的卵子注入Cas9 mRNA获得半克隆胚胎。
  14. 如权利要求13所述的构建基因改造半克隆动物文库的方法，其特征在于，所述表达Cas9的质粒为pX330-mCherry质粒。
  15. 如权利要求12所述的构建基因改造半克隆动物文库的方法，其特征在于，所述半克隆动物文库中的动物为杂合子和/或双等位基因突变的动物。
  16. 如权利要求12所述的构建基因改造半克隆动物文库的方法，其特征在于，所述半 克隆动物文库为非人哺乳动物文库，较优选为啮齿动物文库，更优选为鼠文库，最优选为小鼠文库。
  17. 一种基因改造半克隆动物文库，由权利要求12-16任一权利要求所述的构建基因改造半克隆动物文库的方法构建获得。
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