Patent Description:
Genome-wide recessive genetic screening is an extremely effective and powerful method of identifying the functions of a gene involved in a given biological process. This strategy has been successfully used in lower organisms such as Saccharomyces cerevisiae and nematodes. In mammals, this functional screening is exceptionally difficult due to the diploidy of the genome (<NPL>). The means of targeting RNA interference (RNAi) at mRNA level has now become an optimal solution for genome-wide loss-of-function genetic screening in mammalian cells. However, this method often cannot effectively inhibit the gene expression, while there is an off-target effect (<NPL>). Recently, the bacterial-derived CRISPR-Cas9 system has been successfully used in the screening of genetic defects of the mouse and human at cellular levels. However, CRISPR-Cas9-mediated genome-wide screening is only used at cellular level, which limits the study exclusively to phenotyping at cellular level. In this regard, as currently done with lower organisms such as yeast and nematodes, it is important to achieve efficient, large-scale loss-of-function screening of a broader range of biological processes in mammalian systems.

The obtaining of mammalian haploid embryonic stem cells (haESCs) (<NPL>; and <NPL>) provides a desirable tool for genetic analysis. The androgenetic haploid embryonic stem cells (AG-haESCs) have a whole genome derived from spermatid, with which the full development of a reconstructed embryo can be achieved by intracytoplasmic AG-haESCs injection (ICAHCI) into mature MII oocytes, thereby obtaining a viable animal subject that is referred to as semi-cloned animal. It is inferred that if semi-cloned mice can be produced with AG-haESCs efficiently and stably by ICAHCI, important genes involved in a particular developmental process can be screened out by using AG-haESCs as fertilizing vectors to carry genome-wide CRISPR-Cas9 knockout library.

However, previous studies have shown that the birth rate of viable, semi-cloned mice is very low (where the birth rate is <NUM>% for half-cloned mice and <NUM>% for semi-cloned rats), while approximately <NUM>% of the semi-cloned mice exhibit a phenotype of retarded developmental, and are died shortly after birth. Furthermore, with the long-term culture of AG-haESCs, the overall birth rate of semi-cloned mice declines rapidly, especially for the additional culture resulting from genetic manipulation.

One of the possible reasons is the abnormal expression of imprinted genes. These imprinted genes that are expressed in a parent-of-origin-specific manner are considered as an important barrier to the development of uniparental embryos, so that the normal growth and development of embryos require both the maternal and paternal genomes. At present, about <NUM> imprinted genes have been identified in mice, most of which are located in a large cluster of genes and are regulated by differentially methylated regions (DMRs) (<NPL>). Consistent with this hypothesis, studies have shown that the imprinting of differentially methylated region (DMR) in which the imprinted gene H19 with inhibited paternal expression located is consistently abnormally erased in both AG-haESCs and growth-arrested, semi-cloned mice (<NPL>). The maternally expressed H19 gene is adjacent to the paternally expressed Igf2, and both of them are regulated by the same DMR. This DMR is amenable to DNA methylation in the paternal allele, and as a CTCF-dependent insulator, permits the expression of the maternal allele only. The methylation state of this DMR determines whether H19 (where H19 is expressed, DMR is demethylated, and the insulator is activated) or Igf2 (where Igf2 is expressed, DMR is methylated, and the insulator is deactivated) is expressed. Interestingly, knockout of the H19 gene or its DMR does not lead to any severe phenotype in mice (<NPL>; and <NPL>). At present, there are related researches on reconstructed embryos obtained by using fully mature oocytes and genetically modified immature oocytes (<NPL>; and <NPL>). However, the immature oocytes cannot be cultured and expanded in vitro, so the in-vitro genetic modification cannot be realized; and the technique of obtaining reconstructed embryos from fully-matured oocytes and genetically modified immature oocytes is difficult in operation, so the application value is not high.

The most possible cause of low birth rate is the occurrence of abnormal imprinting status of the androgenetic haploid. It is surprisingly found through experiments that characteristics of the AG-haESCs resembling those of a round spermatid can be established by knocking out the differentially methylated regions (DMRs) of two imprinted clusters H19-Igf2 and Dlk1-Dio3. Such AG-haESCs with two DMRs knocked out is designated as DKO-AG-haESCs. With the DKO-AG-haESCs, semi-cloned mice can be obtained effectively (with a birth rate of SC mice of about <NUM>%), and semi-cloned mouse animals with multiple genetic modifications can still be produced stably after in-vitro genetic manipulation of the AG-haESCs. Unexpectedly, a large number of mutant animals can be effectively obtained in one step simply by transfecting the DKO-AG-haESCs with stable expression of sgRNA library and Cas9 in vitro at the cellular level. These experimental results show that DKO-AG-haESCs can be used as an intermediate to achieve gene mutation at the individual level by carrying the sgRNA library and therefore can be further used in gene mutation-based large-scale screening at individual animal level.

A first aspect of the present invention provides a method for constructing a genetically modified semi-cloned mouse animal, which comprises combining an AG-haESC in which H19 DMR and IG-DMR genes are both knocked out with an oocyte to obtain a semi-cloned embryo, and incubating the semi-cloned embryo, to obtain a semi-cloned mouse animal.

A second aspect of the present invention provides a genetically modified animal, which is constructed according to the above method.

A third aspect of the present invention provides a method for constructing a genetically modified semi-cloned mouse animal library with the AG-haESCs according to the present invention and a lentiviral sgRNA library.

A fourth aspect of the present invention provides a genetically modified semi-cloned mouse animal library constructed by using the above method.

The present invention has the following beneficial effects.

The present invention provides a method for constructing a genetically modified semi-cloned mouse animal, which comprises: combining an AG-haESC in which H19 DMR and IG-DMR are both knocked out with an oocyte to obtain a semi-cloned embryo, and incubating the semi-cloned embryo to obtain a semi-cloned mouse animal.

The AG-haESCs have a whole genome derived from spermatid, has the self-replication ability and pluripotency of stem cells, and can replace the spermatid to combine with oocytes to support the full development of embryos.

H19 DMR refers to a differentially methylated region (DMR) within the H19-Igf2 imprinted cluster. The specific location and sequence of H19 DMR can be determined according to the existing methods such as methylation sequencing or homologous sequence analysis and prediction. Human H19DMR is known to be located in the lpl5. <NUM> region of the chromosome <NUM> and the mouse H19 DMR is located at the distal end of chromosome <NUM> between the two genes H19 and Igf2, a position from 2kb to 4kb upstream of the H19 gene. H19 DMR is methylated on the paternal allele, resulting in the inability of CTCF protein to bind to this methylated region so that the enhancer downstream of H19 does not need to cross over the obstacle CTCF, thereby increasing the expression of upstream Igf2 and decreasing the H19 expression. H19 DMR is demethylated on the maternal allele, and the CTCF protein is able to bind to this unmethylated region, so the enhancer downstream of H19 can only increase the H19 expression, and cannot regulate the upstream Igf2. If the paternal H19 DMR is knocked out, the enhancer downstream of H19 can upregulate the expression of Igf2. Since the androgenetic haploid is of paternal origin, theoretically it should be in a completely methylated state. However, studies show that the H19 DMR in the androgenetic haploid cultured in vitro suffers from abnormally erased methylation, and becomes demethylated, so that the expression of H19 is abnormally up-regulated and the expression of Igf2 is down-regulated. In the present invention, H19 DMR is knocked out and the abnormal state in which H19 expression is up-regulated and the Igf2 expression is down-regulated is corrected.

IG-DMR refers to a differentially methylated region (DMR) within the Dlk-Dio3 imprinted cluster. The specific location and sequence of IG DMR can be determined according to the existing methods such as methylation sequencing or homologous sequence analysis and prediction. The mouse IG-DMR is known to be located on the chromosome <NUM> in a <NUM>. 15kb repeat between the genes Dlk1 and Gtl2 in the imprinted cluster, and the human IG-DMR is located on the chromosome <NUM> (14q32. IG-DMR is DNA methylated on the paternal allele, so the gene Gtl2 and some micromRNAs in this imprinted cluster are not expressed while the gene Rtl1, Dlk1 and Dio3 are expressed. IG-DMR is un-DNA methylated (in demethylated state) on the maternal allele, so Gtl2 and some micromRNAs are expressed while the gene Rtl1, Dlk1 and Dio3 are not expressed. In the androgenetic haploid (of paternal origin) and SC animals born abnormal, studies show that the normally methylated IG-DMR suffers from abnormally erased methylation, causing the silencing of the genes Rtl1, Dlk1, and Dio3, and the abnormal activation of Gtl2 and some microRNAs.

Further, the AG-haESCs undergo other genetic modifications in addition to H19 DMR and IG-DMR knockouts.

Specifically, genetic modification refers to the structural change of a gene made by a biological, chemical or physical means compared with that before modification, and this change mainly refers to the change in base pair composition, comprising, but not limited to, changes caused by the replacement, insertion, and deletion of one or more base pairs.

Genetic modifications of Tetl, Tet2, Tet3 and p53 family of genes are exemplified but are not part of the invention.

Compared with AG-haESCs in which H19 DMR and IG-DMR are not both knocked out, the birth rate of semi-cloned mouse animals constructed with the AG-haESCs of the present invention is higher.

The H19 DMR and IG-DMR can be knocked out by using an existing gene editing method. In a preferred embodiment, the H19 DMR and IG-DMR are knocked out using CRISPR/Cas9-mediated gene manipulation. Gene knockouts may also be performed by other methods, and the present invention is not limited to the methods listed in the examples.

Due to the H19 DMR knockout, the complete sequence of H19 DMR is removed from the chromosome DNA; and due to the IG DMR knockout, the complete sequence of IG-DMR is removed from the chromosome DNA.

In an embodiment, H19 DMR knockout AG-haESCs are constructed firstly, and then IG-DMR is further knocked out. In another embodiment, IG-DMR knockout AG-haESCs are constructed firstly and then H19 DMR is further knocked out. In another example, AG-haESCs in which H19 DMR and IG-DMR are both knocked out are directly constructed.

Further, the AG-haESCs also undergo other genetic modifications.

Such other genetic modifications refer to genetic modifications other than H19 DMR and IG-DMR knockouts. Such other genetic modifications may be the modification of a single target gene or the modifications of multiple target genes of interest. The target gene of interest is not specific and can be set and modified as desired in the research. For example, such other genetic modifications may be the modifications of one, two or more target genes. The AG-haESCs of the present invention in which H19 DMR and IG-DMR are both knocked out can be passaged in vitro, and thus they can theoretically be genetically modified constantly. The number of modifications made to the target gene can be set as needed without particular limitation.

The genetic modifications comprise, but are not limited to, knock-in and knock-out of a target gene, and the like. The knock-in and knock-out of a target gene may be accomplished by techniques such as gene targeting and homologous recombination, comprising, but not limited to, genetic manipulation based on ZFN (zinc finger nuclease), TALEN (transcriptional activator-like effector nuclease) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats).

In an embodiment, the AG-haESCs in which the H19 DMR and IG-DMR are both knocked out undergo one or more genetic modifications to obtain the AG-haESCs with H19 DMR and IG-DMR knockouts and with other genetic modifications. Alternatively, AG-haESCs can be genetically modified first, followed by knocking out H19DMR and IG-DMR from the genetically mutated AG-haESCs. In addition to the above, other biotechnological means that can achieve the H19 DMR and IG-DMR knockouts and other genetic mutations can also be used to construct the AG-haESCs with H19 DMR and IG-DMR knockouts and with other genetic modifications.

In general, the oocytes and the AG-haESCs are derived from the same kind of animal, preferably the same species of animal.

The semi-cloned embryo may specifically be a semi-cloned embryo obtained by ICAHCI using the AG-haESCs in which H19 DMR and IG-DMR are both knocked out as a donor for ICAHCI.

Further, the semi-cloned mouse animal can be obtained by incubating the semi-cloned embryo in a suitable female organism by embryo transfer. In a preferred embodiment, the suitable maternal is a pseudopregnant ICR female rat.

Further, the AG-haESCs undergoes other genetic modifications.

The present invention further provides a genetically modified animal, which is constructed according to the above method.

The present invention also provides a method for constructing a genetically modified semi-cloned mouse animal library, which comprises the steps of.

The sgRNA lentiviral library plasmids comprise several lentiviral vectors that express different sgRNAs. The lentiviral sgRNA library can be constructed by current technologies, or existing lentiviral sgRNA library plasmids may be used. Specifically, sgRNAs designed for different genes can be cloned into lentiviral vectors. The sgRNA can be designed according to the gene of interest.

In a preferred embodiment, a commercially available lentiviral sgRNA library of whole genomes of mice is employed.

Specifically, Step <NUM>) may be selected from any one of:.

The AG-haESC library carrying the sgRNA library is further transfected with a plasmid expressing Cas9, and the semi-cloned embryos are obtained by ICAHCI using the resultant AG-haESCs as a donor for ICAHCI.

AG-haESCs in the AG-haESC library carrying the sgRNA library, as a donor for ICAHCI, are injected into mature oocytes by ICAHCI, and then Cas9 mRNA is injected into the reconstructed oocytes, to obtain the semi-cloned embryos.

The AG-haESC library carrying the sgRNA library is further transfected with a plasmid expressing Cas9, the resultant AG-haESCs, as a donor for ICAHCI, are injected into mature oocytes by ICAHCI, and then Cas9 mRNA is injected into the reconstructed oocytes, to obtain the semi-cloned embryo, from which a semi-cloned mouse animal is obtained after embryo transfer.

In the methods A and C, the plasmid expressing Cas9 can be constructed by cloning the Cas9 expressing gene into an expression plasmid. In a preferred embodiment, the plasmid expressing Cas9 is pX330-mCherry plasmid. The plasmid that is constructed to express Cas9 is not limited to the pX330 plasmid. The expression plasmid only needs to be suitable for expression of exogenous genes in mammalian cells.

The present invention further provides another method for constructing a genetically modified semi-cloned mouse animal library, which comprises the steps of:.

The virus particles expressing Cas9 can be obtained by cloning the encoding gene expressing Cas9 into a lentiviral vector and then packaging the lentivirus in the prior art. The lentiviral vector expressing Cas9 is commercially available.

The semi-cloned mouse animal library comprises several genetically mutated semi-cloned mouse animals. The animals may be heterozygous or biallelic mutant animals.

Further, semi-cloned mouse animals can be obtained by culturing the semi-cloned embryos in a suitable female organism by embryo transfer. In a preferred embodiment, the suitable female organism may be a pseudopregnant ICR female rat.

The present invention also provides a genetically modified semi-cloned mouse animal library, which is constructed according to the method as described above.

The genetically modified semi-cloned animal library of the present invention can be used in genetic screening of genes at subordinate individual level.

The embodiments of the present invention are described below with reference to specific examples.

When a numerical range is given in an example, it is to be understood that both endpoints of each numerical range and any numerical value between the two endpoints are encompassed, unless the context otherwise indicates. Unless defined otherwise, all technical and scientific terms as used herein have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment and materials used in the examples, the present invention may be implemented using any of the methods, devices, and materials in the prior art that are similar or equivalent to the methods, devices, and materials described in the examples of the present invention, based on the knowledge of those skilled in the art the prior art and the disclosure of the present invention,.

Unless otherwise specified, in the experimental methods, detection methods, and preparation methods disclosed in the present invention, the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and conventional techniques in related fields are adopted. These techniques are well documented in the literature.

The cell culture medium (DMEM), fetal bovine serum (FBS), serum replacement (KSR), trypsin, Opti-MEM, DPBS, and Lipofectamine <NUM> were purchased from Life Technologies Inc. ; the restriction endonucleases and T4 ligase were purchased from NEB; the Taq enzyme and dNTPs were purchased from TaKaRa; the CDNA reverse transcription kit, and fluorescent quantification reagent SYBR-Green were purchased from TOYOBO; and the oligonucleotides were synthesized by Shanghai Generay Company.

H-CZB Stock <NUM>, Hepes. 2Na (sigma, CAT#H0763) or ICN <NUM> or Hepes (sigma, CAT#H4034) <NUM>, NHCO<NUM> <NUM>, CaCl<NUM>·<NUM><NUM>O 100x stock <NUM>, Pyruvate <NUM>, and Glutamin 200x stock <NUM>, adjusted to pH <NUM>, and filtered well.

CZB stock <NUM>, CaCl<NUM>•<NUM><NUM>O 100x stock <NUM>, Pyruvate <NUM>, Glutamin 200x stock <NUM>, BSA <NUM>.

enzymatically cleaving px330 (addgene) plasmid with NotI, and then inserting a CMV-mcherry-pA fragment amplified from pmCherry-C1 (Clontech) into the enzymatically cleaved px330 plasmid.

The mouse testis was digested with collagenase IV for <NUM> minutes and then with trypsin for <NUM> minutes, and then sorted by FACS, to obtain round spermatid of mice.

All animals were used in accordance with the procedures in the animal operation manual of Institute of Biochemistry and Cells, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

H19 Δ3.8kb KO mice (C57/B6 background, homozygous): constructed as described in (<NPL>.

IG-DMRKO mice (C57/B6 background, heterozygous): constructed as described in (<NPL>,).

B6D2F1 (C57BL/<NUM> X DBA2) female mice: female offspring obtained after mating with female mice of C57BL/<NUM> strain with male mice of DBA2 strain.

Pseudopregnant ICR female mice: ICR adult female mice purchased from SLAC Laboratory Animal Co. , Ltd were mated with ligated adult ICR male mice, to obtain pseudopregnant ICR female rats.

The AG-haESC line was constructed according a reported method (<NPL>).

The MII oocytes were enucleated, into which the corresponding sperm heads were injected. Mouse MII oocytes were harvested <NUM> hours after treatment with human chorionic gonadotropin (HCG) and then enucleated using a Piezo needle in HEPES-CZB medium containing <NUM>µg/ml cytochalasin B (CB). After enucleation, single sperm heads were injected into the cytoplasm of the oocytes. The reconstructed embryos were cultured in CZB medium for <NUM> hour and then transferred to the activation solution containing <NUM> Sr<NUM>+ for activation. After activation, all reconstructed embryos were transferred to KSOM medium containing amino acids and incubated at <NUM>, and <NUM>% CO<NUM>. The reconstructed embryos reaching the morula or blastula stage <NUM> days later were seeded in ESC medium.

The zona pellucid of the reconstructed embryos was removed by digestion with the acid Tyrode solution. Each embryo was transferred to a <NUM>-well plate plated with a mouse fibroblast feeder layer and cultured with ESC medium containing <NUM>% serum replacement (KSR), <NUM>,<NUM> U/ml LIF, <NUM> CHIR99021 and <NUM> PD0325901. After <NUM>-<NUM> days of culture, the cell clones were trypsinized and transferred to a <NUM>-well plate plated with a fresh feeder layer. The cells were further expanded, and passaged into a <NUM>-well plate and further into a <NUM>-well plate, and the cells were daily maintained in a <NUM>-well plate. To sort the haploid cells, after the embryonic stem cells were trypsinized, they were washed once with PBS (GIBCO) and then in ESC medium containing <NUM>µg/ml Hoechst <NUM>. After being placed in a water bath for <NUM>, haploid cells of 1N peak were sorted by the flow cytometer BD FACS Ariall and subsequently subcultured to obtain the AG-haESC line.

Construction of CRISPR-Cas9 plasmid: The synthesized forward oligonucleotide strand and reverse oligonucleotide strand of sgRNA were annealed to obtain a double-stranded oligonucleotide strand (in the present invention, the sgRNA sequence refers to the sequence of the forward oligonucleotide strand of the sgRNA), which was then ligated to pX330-mCherry enzymatically cleaved with BbsI (New England Biolabs). The constructed corresponding plasmid was transfected into the AG-haESCs using Lipofectamine <NUM> (Life Technologies) according to the instructions. <NUM> hours after transfection, the haploid cells with the red fluorescent protein were sorted by flow cytometry (FACSAriaII, BD Biosciences) and then plated at a low density. After <NUM>-<NUM> days of growth, monoclones were picked up for subsequent construction of cell lineages. Finally, cell lines with corresponding gene mutations were obtained by sequencing target genes by PCR.

If gene knock-in was involved, a double-stranded DNA donor needed to be constructed.

A sequence encoding EGFP, mCherry or ECFP was amplified and then ligated to the pMD19-T vector, to give pMD19-T-EGFP/mCherry/ECFP. Subsequently, the left and right homologous arms of the target gene were inserted into the pMD <NUM>-T-EGFP/mCherry/ECFP vector.

The viral sgRNA library of mice and the Cas9 expressing viral plasmid have been reported mice (<NPL>; and <NPL>). The viral sgRNA library of mice and the Cas9 expressing viral plasmid used in the present invention were provided by Addgene. To prepare the virus, HEK293T was passaged in advance into a <NUM> petri dish, <NUM>µg of viral plasmid (lentiviral sgRNA library or lentiviral Cas9) and <NUM>µg of ViraPower Lentiviral Packaging Mix (Invitrogen) were transfected into HEK293T cells by using Lipofectamine® <NUM> Reagent (Invitrogen, Life Technologies). The supernatant were collected <NUM> hours after transfection and concentrated with Lenti-Concentin virus precipitation solution (SBI) and then stored at -<NUM>.

Infection with lentiviral Cas9: A cell suspension of <NUM><NUM> DKO-AG-haESCs was infected for <NUM> hours with <NUM>µg/ml polybrene (Sigma) and packaged lentiviral Cas9, and then screened for <NUM> days with <NUM>µg/ml blasticidin (Sigma). The remaining resistant clone was a cell line integrated with lentiviral Cas9.

Infection with lentiviral sgRNA library: A cell suspension of <NUM><NUM> DKO-AG-haESCs was infected for <NUM> hrs with <NUM>µg/ml polybrene (Sigma) and packaged lentiviral CRISPR-sgRNA library, and then screened for <NUM> days in a medium comprising <NUM>µg/ml puromycin (Invitrogen), to obtain a positive clone that is a cell line carrying lentiviral sgRNA library.

A cell line integrated with lentiviral Cas9 was prepared first, then further infected with the lentiviral sgRNA library, and then screened for <NUM> days in a medium comprising <NUM>µg/ml puromycin (Invitrogen), to obtain a positive clone that is a cell line integrated with lentiviral Cas9 and lentiviral sgRNA library.

If the DKO-AG-haESC cell line was only infected with the lentiviral sgRNA library, then the cells did not express Cas9. At this time, transfection with the pX330-mCherry plasmid at the cellular level was needed to achieve the genome editing.

If the EZ DNA methylation Gold kit (ZYMO Research) was used, an appropriate amount of DNA was prepared, and the following procedures were operated according to the steps of use of the kit. The resulting product recovered with the kit was used as a template for PCR, the product was recovered and then ligated to the PMD19-T vector, followed by transformation and plating. <NUM> colonies were picked from the plate for sequencing.

Total RNA was extracted from the cells or organs with Trizol reagent (Invitrogen) and then <NUM>µg of total RNA was reversely transcripted into cDNA using the First Strand cDNA Synthesis kit (TOYOBO). Real-time fluorescent quantitative PCR reactions were performed on a Bio-Rad CFX96 instrument using SYBR Green Realtime PCR Master Mix (TOYOBO), with <NUM> replicates for each set of samples. All the gene expression levels were detected with the expression level of housekeeping gene Gapdh as an internal reference.

To obtain semi-cloned (SC) embryos, AG-haESCs were treated for <NUM> hrs with a medium containing <NUM>µg/ml colchicine to synchronize the cells to M phase and then intracytoplasmically injected into the oocytes. The digested AG-haESCs were washed <NUM> times with HEPES-CZB medium and then re-suspended in <NUM>% (w/v) polyvinylpyrrolidone (PVP) in HEPES-CZB medium. Nuclei of AG-haESCs in M phase were injected into MII oocytes under a Piezo microscope. The reconstructed embryos were cultured in CZB medium for <NUM> hour and then activated with a CB-free medium for <NUM>-<NUM> hours. After activation, all the reconstructed embryos were cultured in KSOM medium at <NUM>, and <NUM>% CO<NUM>. ICAHCI embryos reached <NUM>-cell embryo after being cultured in the KSOM medium for <NUM> hours.

The operation followed a reported method (<NPL>).

Every <NUM>-<NUM><NUM>-cell embryos obtained by ICAHCI or ROSI were transferred into each uterus of pseudopregnant ICR mice at <NUM> dpc (<NUM> day post-mating). The female mice experienced caesarean section or natural birth after <NUM> days of pregnancy. Caesarean section was done for reconstructed embryos obtained with WT AG-haESCs or single DMR knockout AG-haESCs, and expired fetuses were quickly peeled off from the female's uterus. For embryos obtained by ROSI or with DKO-AG-haECs, females after <NUM> days of pregnancy experienced natural birth. After removing the fluid from the born mice, the mice were placed in an oxygen incubator, and survived mice were subsequently nourished by the surrogate females.

The RNA-seq library of total RNA was established according to Illumina's official TreSeq RNA Sample Prep v2 Guide. After establishment, deep sequencing was performed on the IlluminaHiSeq <NUM> instrument available from the Computational Biology Center of the Institute of Computing Biology, Chinese Academy of Sciences-Max-Planck-Gesellschaft zur Förderung der Wissenschaften e. <NUM> samples, comprising <NUM> WT AG-haESCs, H19△DMR - IG△DMR _AGH, H19△DMR-IG△DMR-AGH-OG3, IG△DMR-H19△DMR-AGH and round spermatid were subjected to subsequent analysis.

The algorithm for gene expression level was RPKM, specifically as described in (<NPL>).

The p-value of differentially expressed genes was calculated using the waldscore method (<NPL>) in which abs (waldscore) was set to be ><NUM> (that is, p-value<<NUM>), and then the differentially expressed genes were screened.

The RRBS library was established according to Illumina's official protocol, and then sequenced on the IlluminaHiSeq <NUM> instrument (<NPL>. All sequencing reads were aligned with the mouse genome.

The extracted genomic DNA was amplified by PCR using corresponding primers, and the PCR product was further subjected to agarose gel electrophoresis.

The haploid sperm heads of H19 Δ3.8kb mice were injected into enucleated oocytes (<FIG>) following the method as described in Section <NUM>, to obtain reconstructed blastocysts, with which AG-haESC cell lines were constructed. Three haploid cell lines were established from <NUM> reconstructed blastocysts (which were designated as H19△DMR-AGH-<NUM>, H19△DMR-AGU-<NUM>, H19△DMR-AGU-<NUM>) (<FIG>).

Following the method as described in Section <NUM>, H19△DMR-AGH cells were used as a donor for ICAHCI, and <NUM><NUM>-cell embryos reconstructed with these <NUM> cell lines were transferred into the pseudopregnant female mice. Finally, <NUM> healthy, viable semi-cloned mice and <NUM> growth-arrested mice were obtained by caesarean section of the female mice on day <NUM> after pregnancy (<FIG>, Table <NUM>). The birth rate in normal half-cloned mice is about <NUM>%; however, about <NUM>% of the semi-cloned mice developed from the embryos constructed with H19△DMR-AGH still have abnormal growth.

Semi-cloned mice were constructed with wild-type AG-haESCs as a donor for ICAHCI. The birth rate of normal semi-cloned mice is about <NUM>-<NUM>%.

Fluorescent quantitative PCR was used to detect whether aberrant high expression of Gtl2 was also present in the major organs of H19△DMR-AGH-derived growth-arrested mice, following the method as described in Section <NUM>.

The experimental results show that Gtl2 overexpression occurs in most of the organs tested in growth-arrested semi-cloned mice compared to normal mice (Fig. S1E).

Experimental Methods: The analysis was carried out following the methods as described in Sections <NUM> and <NUM>.

The results of methylation analysis show that the growth-arrested semi-cloned mice show obvious hypomethylation in the IG-DMR region of the differential methylation sites in the Dlk1-Gtl2 imprinted cluster (Figs. Interestingly, a more severe loss of IG-DMR methylation imprinting occurred in the later generation of H19△DMR-AGH (H19△DMR-AGU-<NUM>, pl6) compared to with its earlier generation (H19△DMR-AGH-<NUM>, p7), resulting in a significantly increased rate of growth arrested mice (Fig. S1H, Table S1). This indicates that the abnormal expression of Gtl2 may be another important factor leading to the failure in development of the semi-cloned mice obtained with H19△DMR-AGH cells.

The construction was carried out following the method as described in Section <NUM>.

<NUM> sgRNAs were designed according to the sequence between Dlk1 and Gtl2 which had a <NUM>. 15kb IG-DMR knocked out (designated as IG-DMR-sgRNA1 and IG-DMR-sgRNA2) (Fig. IF).

The plasmids pX330-mCherry expressing Cas9 and IG-DMR-sgRNAs were constructed and transfected into the H19△DMR-AGH cells, to finally obtain <NUM> AG-haESC lines. The sequencing of the PCR product of the target gene (IG-DMR deletion check-F: TGTGCAGCAGCAAAGCTAAG; IG-DMR deletion check-R: ATACGATACGGCAACCAACG) found that the IG-DMR were successfully knocked out in <NUM> cell lines (designated as H19△DMR- IG△DMR _AGH-<NUM> through H19△DMR - IG△DMR-AGH-<NUM>) (<FIG>, S2A and S2B). Off-target analysis of H19△DMR-IG△DMR-AGH-<NUM> shows that no mutations occur to a total of <NUM> potential off-target sites (Table S2), where these sites are predicted by genome-wide searching by using software reported previously (<NPL>).

Following the method as described in Section <NUM>, H19△DMR-IG△DMR-AGH cells were used as a donor for ICAHCI to construct semi-cloned mice. The results show that <NUM>% of the semi-cloned embryos can well developed (<FIG>, Fig. S2C, Table <NUM>, Table S1), which is similar to the ROSI birth rate reported by our laboratory (Table <NUM>) or other laboratories (<NPL>. Furthermore, compared with the semi-cloned mice obtained with wild type AG-haESCs and H19△DMR-IG△DMR-AGH by ICAHCI that require caesarean section, after the embryos reconstructed with H19△DMR-IG△DMR-AGH-<NUM> is transferred to pseudopregnant female mice, the pseudopregnant female mice can carry out natural birth, and the born semi-cloned mice all survive healthy. The data suggests that H19△DMR-IG△DMR-AGH cells obtain characteristics resembling a round spermatid successfully by knocking out IG-DMR, from which semi-cloned mice can be produced efficiently.

Following the method as described in Section <NUM>, IG△DMR-AGH cells were used as a donor for ICAHC to construct semi-cloned mice. It is found that the IG△DMR-AGH cells are not donors effective in the production of semi-cloned mice (Table <NUM>, Table S1), with which it is difficult to obtain healthy normal SC mice (where only <NUM> normal SC mice are obtained from <NUM> transferred embryos) (<FIG> and S2E). Moreover, most of the mice are growth arrested.

The result show that the H19 DMR of IG△DMR-AGH cell line suffers erased methylation, and complete loss of methylation occurs in the growth-arrested mice (<FIG>, S2F, and S2G).

The H19 Δ3.8kb DMR were knocked out from the IG△DMR-AGH cell line obtained in the above example by the CRISPR-Cas9 method (see <NPL>), and <NUM> DKO-AG-haESC cell lines were successfully obtained (designated as IG△DMR-H19△DMR-AGH-<NUM> through IG△DMR-H19△DMR-AGH-<NUM>) (<FIG> and S2H, and Table S2).

Following the method as described in Section <NUM>, <NUM> cell lines above were used as a donor for ICAHCI to construct semi-cloned mice. The results show that the <NUM> cell lines have the similar ability to produce health SC mice as the H19△DMR-IG△DMR-AGH cells (Figs. S2I and 2J, and Table <NUM>, Table S1).

It has been reported that the <NUM>nd generation of this cell line has substantially lost the ability to produce healthy, semi-cloned mice (<NPL>).

The oligo of sgRNA were annealed and then the sgRNAs of H19 and IG-DMR were respectively ligated to the BbsI digested px330-mCherry plasmid and transformed. Plasmid was extracted from the bacterial suspension sequenced to be correct for subsequent transfection.

The 21st generation of the AGH-OG-<NUM> cell line was transformed with the plasmid obtained above.

<NUM> H19 and Gtl2 DMR double knockout AG-haESC cell lines (designated as H19△DMR-IG△DMR-AGH-OG3-<NUM> through H19△DMR-IG△DMR-AGH-OG3-<NUM>) were finally obtained (<FIG> and S3A and Table S2).

Following the method as described in Section <NUM>, H19△DMR-IGL△DMR-AGH-OG3 cells were used as a donor for ICAHCI to construct semi-cloned mice. Surprisingly, by injecting <NUM> of these cell lines into MII oocytes, about <NUM>% of semi-cloned embryos were able to develop fully (Fig. S3B, S3C, Table <NUM>, and Table S1). This shows that WT-AG-haESCs, which had previously completely lost the ability to produce SC mice, were able to regain characteristics resembling a round spermatid after knockdown of H19-DMR and IG-DMR.

Three H19-DMR and IG-DMR double-knockout AG-haESC cell lines were constructed by using the three methods described in the foregoing Examples <NUM>, <NUM> and <NUM>, and a total of <NUM> SC mice were obtained. A mouse birth rate of <NUM>% was achieved with the transferred embryos. These DKO-AG-haESC derived SC mice are able to grow to adulthood and are capable of producing offspring. Genotyping of <NUM> neonatal mouse offspring in <NUM> litters find that <NUM> animals carry H19-DMR knockout and <NUM> animals are WT (<FIG>). <NUM> out of the other <NUM> mice have IG-DMR knockout and <NUM> have H19 and Gtl2 DMR double knockouts. The <NUM> mice died shortly after birth, consistent with the previously reported lethal phenotype of the maternally inherited IG-DMR before or after birth (<NPL>).

RNA-seq and gene expression analysis were performed following the methods described previously in Sections <NUM> and <NUM>.

Genome-wide methylation level analysis was performed following the method as described in Section <NUM>.

The results of Q-PCR showed that the expression of H19 and Gtl2 is down-regulated while the expression of Igf2 and Dlk1 is up-regulated in DKO-AG-haESCs (Fig. S3D). The gene expression profiles of DKO-AG-haESCs, normal AG-haESCs and round spermatids were compared. Clustering data based on RNA-seq reveals that the expression profile of DKO-AG-haESCs is highly similar to that of WT-AG-haESCs, but greatly different from that of round spermatids (<FIG>, S3E). Further analysis of the expression profiles of imprinted genes in DKO-AG-haESCs and WT-AG-haESCs revealed that all imprinted genes in DKO-AG-haESCs and WT-AG-haESCs have extremely similar expression levels (<FIG>). In order to further evaluate the epigenetics, reduced representation bisulfate sequencing (RRBS) was performed to detect the genome-wide methylation level. As shown in <FIG>, S3F, and S3G, knockouts of H19 and Gtl2 DMRs do not alter the methylation levels in the promoter regions of all the genes and imprinted genes detected. Taken together, the results show that H19 and Gtl2 DMRs are two major impairments for AG-haESCs in obtaining characteristics resembling spherical spermatid.

Production of semi-cloned mice with DKO-AG-haESCs carrying multiple genetic modifications.

Initial cells: DKO-AG-haESCs. Knockouts of the TET family of genes employed the DKO-AG-haESCs prepared in Example <NUM>, and knockouts of the p53 family of genes employed the DKO-AG-haESCs prepared in Example <NUM>.

Target mutations: mutations of Tet1, Tet2, Tet3, and p53 family.

sgRNAs of Tet1, Tet2, and Tet3 were annealed respectively, and ligated to a BbsI digested px330-mCherry plasmid respectively, and positive plasmids in which sgRNAs were ligated were picked up by sequencing.

The plasmids expressing the sgRNAs of Tet1, Tet2, and Tet3 were co-transformed into the DKO-AG-haESC cell line. mCherry positive cells were sorted, and plated in a Petri dish. After <NUM> days of growth, the clones were picked and passaged for amplification. The established cell line was identified for the Tet1, Tet2, and Tet3 mutations by sequencing the PCR product.

sgRNAs of p53, p63, and p73 were annealed respectively, and ligated to a BbsI digested px330-mCherry plasmid respectively, and positive plasmids in which sgRNAs were ligated were picked up by sequencing.

The plasmids expressing the sgRNAs of p53, p63, and p73 were co-transformed into the DKO-AG-haESCs cell line. mCherry positive cells were sorted, and plated in a Petri dish. After <NUM> days of growth, the clones were picked and passaged for amplification. The established cell line was identified for the p53, p63, and p73 mutations by sequencing the PCR product.

Following the method as described in Section <NUM>, the cells constructed above were used as a donor for ICAHCI to construct semi-cloned mice.

In this example, Tet1, Tet2 and Tet3 were mutated in DKO-AG-haESCs by CRISPR-Cas9 method. The plasmids constructed to express Cas9 and <NUM> sgRNAs of Tetl, <NUM>, and <NUM> (<FIG>) (see <NPL>) were transformed into DKO-AG-haESCs. <NUM> DKO-AG-haESC lines were finally established. The sequencing of the PCR products of the Tetl, <NUM>, and <NUM> gene revealed that triple gene mutations occur in <NUM> cell lines (designated as Tet-TKO-DAH-<NUM> through Tet-TKO-DAH-<NUM>) (<FIG> and <FIG>). ICAHCI results of <NUM> of the cell lines indicated that SC mice with Tet1, <NUM> and <NUM> mutations can be obtained with corresponding rate (<FIG> and S4B, Table <NUM> and Table S3). Corresponding SC mice can also be obtained with DKO-AG-haESCs with mutant p53 family of genes by ICAHCI (Figs. S4D, S4E, Table <NUM> and Table S3). These results show that WT-AG-haESCs are failed to produce viable SC mice after in vitro genetic manipulation, but DKO-AG-haESCs are still able to efficiently and stably produce semi-cloned mice after gene editing.

Gene editing of DKO-AG-haESCs and production of semi-cloned mice therewith.

The <NUM> plasmids, comprising plasmids carrying Tetl and Tet3 sgRNA, and Tet1-EGFP and Tet3-ECFP donors were co-transfected into the DKO-AG-haESCs cell line. mCherry positive cells were sorted, and plated in a Petri dish. After <NUM> days of growth, the clones were picked and passaged for amplification. The established cell line was identified for the Tet1-EGFP and Tet3-ECFP knockins by PCR. The double knock-in cells were designated as Tet1&<NUM>-KI-DAH.

sgRNA of Tet2 was annealed and ligated to a BbsI digested px330-mCherry plasmid, and a positive plasmid in which sgRNA was ligated was picked up by sequencing.

For the preparation of Tet2-mCherry donor, by using pmCherry-N1 as a template, and P2A-fluorescence F: GCCACGAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGTGAGCAAGGGCG AGGAG and P2A-fluorescence R: CTTGTACAGCTCGTCCATG as primers, a sequence encoding mCherry was amplified and then ligated to a pMD19-T carrier. Subsequently, the left and right homologous arms of the gene of interest were respectively inserted into two sides of the mCherry sequence in the pMD19-T-mCherry vector.

The <NUM> plasmids, comprising the plasmid carrying Tet2 sgRNA and the Tet2- mCherry donor were co-transfected into the Tet1&<NUM>-KI-DAH cell line. mCherry positive cells were sorted, and plated in a Petri dish. After <NUM> days of growth, the clones were picked and passaged for amplification. The established cell line was identified for the Tet2-mCherry knock-in by PCR. The positive cell clone was the Tet-TKI-DAH cell line.

In this example, DKO-AG-haESCs were obtained in which endogenous Tet1, Tet2 and Tet3 were knocked in different fluorescent reporter groups. First, DKO-AG-haESCs were transfected with a plasmid co-expressing Cas9 and Tet1 and Tet3 sgRNAs (<FIG>) and a double stranded DNA donor vector having EGFP and ECFP reporter groups fused respectively to the last terminator of Tet1 and Tet3 (<FIG> and S5A). A total of <NUM> DKO-AG-haESC cell lines were obtained, with <NUM> Tetl-EGFP knock-in and <NUM> Tet3-ECFP knock-in cell lines (Figs. S5B and S5C). One of the cell lines carrying both Tetl-EGFP and <NUM> Tet3-ECFP knock-ins was designated as Tet1 & <NUM>-KI-DAH-<NUM> (Figs. S5D and S5E). ICAHCI results showed that DKO-AG-haESCs carrying Tetl-EGFP and Tet3-ECFP knock-ins have similar semi-cloned mice production capability to WT DKO-AG-haESCs (Figs. S5F and S5G, Table <NUM> and Table S3). Next, the plasmid expressing Cas9 and Tet2 sgRNA and the double-stranded DNA donor vector having the mCherry reporter group fused to the last terminator of the Tet2 gene were transfected into the Tet1 & <NUM>-KI-DAH-l cell line (<FIG> and <FIG>). Out of the established <NUM> cell lines, <NUM> red-KFP knock-in haploid cell lines were identified (designated as Tet-TKI-DAH-<NUM> through Tet-TKI-DAH-<NUM>) (Figs. <NUM> and <FIG>). Finally, the developmental potential of the Tet-TKI-DAH cell line was verified by ICAHCI. Consistently, Tet-TKI-DAH was able to efficiently produce viable semi-cloned mice by injection into oocytes (Figs. S5S, S5J, Table <NUM>, and Table S3). Taken together, these results indicate that it is a possible route to produce SC mice having corresponding genetic characteristics by ICAHCI after multi-gene genetic manipulation of DKO-AG-haESCs.

Large-scale production of heterozygous mutant SC mice with DKO-AG-haESCs carrying sgRNA library.

Following the method as described in Section <NUM> , the virus was prepared and DKO-AG-haESCs were infected with a genome-wide lentiviral sgRNA library, and then transfected with a pX330-mCherry plasmid expressing Cas9, to finally construct DKO-AG-haESCs carrying the sgRNA library.

Following the method as described in Section <NUM>, semi-cloned mice were constructed using the previously constructed cells as a donor for ICAHCI.

The sgRNAs were detected by PCR amplification using specific primers, and then the PCR product was subjected to agar gel electrophoresis to observe the presence of a strip.

According to the sgRNA sequencing, the corresponding gene inserted was identified, and then the upstream and downstream primers were designed by the conventional method in the vicinity of the target gene sgRNA, followed by PCR and sequencing.

At present, the genome-wide sgRNA library has been successfully established and used in the loss-of-function genetic screening in human and mouse cells (<NPL>). This experiment demonstrates that DKO-AG-haESCs are capable of carrying the sgRNA library and that a large number of mutant mouse models can be obtained simply by a one-step method by ICAHCI (<FIG>) (this method is known as Lenti-sgRNA + pX330). In this test, a newly established and more definite mouse lentiviral library was used. This library had <NUM>,<NUM> sgRNAs designed for the genes of <NUM>,<NUM> encoded proteins in mice. <NUM> × <NUM><NUM> haploid cell line IG△DMR-H19△DMR-AGH-<NUM> enriched by FACS were infected with the genome-wide lentiviral sgRNA library. After <NUM> days, these infected cells were treated with puromycin for <NUM> days, followed by transfection with the pX330-mCherry plasmid expressing Cas9. Haploid cells expressing mCherry indicated the successful transfection and expression of Cas9, which were then used in ICAHCI experiments following FACS enrichment (<FIG>). To detect whether the haploid gene is induced to be mutated by CRISPR-Cas9, <NUM> haploid cell clones were randomly selected. All of the detected cell clones carried one sgRNA, which was also found to be mutated by DNA sequencing, indicating that gene mutations can be successfully induced in haploid cells by CRISPR-Cas9.

<NUM> semi-cloned mice were obtained by three independent ICAHCI experiments (<FIG> and Table <NUM>), of which <NUM> carried one sgRNA (<FIG>). Sequencing of the PCR products of sgRNA revealed that <NUM> SC mice harbored one allelic mutation of the gene of interest, <NUM> of which carried insertions or knockouts mutation (indel) that result in frameshift, leading to the loss of function of one allele (<FIG>). Interestingly, all the mutations were of the same genotype, indicating that gene mutations are realized in haploid cells after the transient expression of Cas9. Although the remaining <NUM> mice carried one sgRNA, no DNA cleavage was shown at the site needed to be mutated. This rate was consistent with that previously observed in human cells (<NPL>) or mouse embryos (<NPL>). This data demonstrates that the introduction of genetic mutations into SC mice can be achieved by ICAHCI of DKO-AG-haESCs harboring the sgRNA library that are transiently transfected to express Cas9, to finally obtain a large number of heterozygous mutant mice in one step.

DKO-AG-haESCs were infected with the lentiviral CRISPR-sgRNA library following the method as described in Section <NUM>, to obtain a cell line carrying the lentiviral sgRNA library.

The nuclei of the haploid cells carrying sgRNA were injected into mature oocytes by ICAHCI, followed by intracytoplasmic injection of Cas9 mRNA into the reconstructed oocyte (where this protocol was known as Lenti-sgRNA + Cas9 injection). Semi-cloned mice were then constructed by embryo transfer.

sgRNA detection in semi-cloned mice: The detection was carried out following the method as described in Example <NUM>.

Detection of allelic mutation: The detection was carried out following the method as described in Example <NUM>. In addition, the PCR product of the target gene was ligated into the pMD19-T vector, and then the clones were picked up and sequenced.

In this experiment, haploid cells carrying sgRNA were injected into mature oocytes by ICAHCI and then Cas9 was injected into the reconstructed oocytes (where this protocol was known as Lenti-sgRNA + Cas9 injection). A total of <NUM> SC mice carrying one sgRNA were born, of which <NUM> had genetic mutations (Table <NUM>). Sequencing of the PCR product of the gene of interest revealed that <NUM> mice had only a monoallelic modification, and other <NUM> mice were biallelic mutant (which is <NUM>% of all born SC mice). Sequencing by TA cloning of the <NUM> biallelic mutant SC mice revealed that approximately <NUM>% of the clones carried the insertion or deletion mutations (Table S4).

Detection of allelic mutation: The detection was carried out following the method as described in Example <NUM>.

In this experiment, the pX330-mCheny plasmid was transiently transfected into the sgRNA-bearing haploid cells, and then the cells were injected into oocytes, followed by injection of Cas9 mRNA (Fig. S6A and S6B) (where this protocol was known as Lenti-sgRNA+pX330+Cas9 injection). A total of <NUM> SC mice carrying one sgRNA were obtained. <NUM> mice carried genetic mutations (Figs. S6C and S6D and Table <NUM>), in which <NUM> of the mutant mice carried biallelic mutations (Fig. S6E) (which was <NUM>% of all the SC mice) and the other <NUM> mice were monoallelic mutant. TA cloning and sequencing results of <NUM> biallelic mutant mice revealed that about <NUM>% of the clones have insertion or deletion mutations (Figs. S6F and S6G and Table S4).

Production of mice harboring sgRNA-mediated biallelic mutations with DKO-AG-haESCs in one step.

Specific construction of cell lines by Lenti-Cas9+lenti-sgRNA method: The construction was carried out following the method "co-infection with lentiviral Cas9 + lentiviral sgRNA library" as described in Section <NUM>.

The cell line integrated with lentiviral Cas9 and lentiviral sgRNA library was further sorted by FACS to enrich the haploid cells for use in subsequent ICAHCI operation.

Following the method as described in Section <NUM>, semi-cloned mice were constructed by ICAHCI using DKO-AG-haESCs with constant expression of Cas9 and sgRNA library as a donor.

In this experiment, DKO-AG-haESCs with constant expression of Cas9 and sgRNA library were obtained by two rounds of screening with drugs. SC mice were then generated by ICAHCI (<FIG>) (where this protocol was known as Lenti-Cas9+lenti-sgRNA). <NUM> (<NUM>%) of the <NUM>,<NUM> embryos reconstructed by ICAHCI were developed fully (<FIG>) and the birth rate was basically similar to that obtained with WT DKO-AG-haESCs or DKO-AG-haESCs carrying different gene modifications (Table <NUM>), indicating that after multiple genetic manipulations, the ability of DKO-AG-haESCs to produce SC mice is not affected. A total of <NUM> SC mice carried one sgRNA and were used for subsequent genotyping (<FIG> and Table <NUM>). The results showed that <NUM> SC mice are genetically mutated, of which <NUM> are biallelic mutant mice (<FIG>). <NUM> monoallelic mutant SC mice were obtained. Although Cas9 in these mice was supposed to be constantly expressed, silencing of the viral vector led to transcriptional silencing of Cas9 at different development stages of the SC mice. TA cloning and sequencing of <NUM> biallelic mutant SC mice showed that about <NUM>% of the clones have insertion or deletion mutations (<FIG>). In order to further determine the mutation efficiency throughout the body of the SC mice, the genetic mutations in different organs (comprising the brain, heart, kidney, liver and lung) of <NUM> SC mice were analyzed and all of these organs were found to have biallelic mutations. Finally, TA cloning and sequencing of different organs in one SC mouse carrying Scube1 mutation was performed. The results showed that about <NUM>% of the clones have insertion or deletion mutations (<FIG>). Interestingly, this SC mouse died within an hour after birth, consistent with previous reports that the Scube1 mutant mouse died shortly after birth (<NPL>). In conclusion, the above experimental results provide sufficient evidence for the possibility of obtaining a large number of mutant mice with DKO-AG-haESCs carrying the sgRNA library, thereby achieving the large-scale gene knockout-based screening at the mouse level, which greatly simplifies the DKO-AG-haESC-mediated gene knockout-based screening.

Claim 1:
A method for constructing a genetically modified semi-cloned mouse animal, comprising: combining an androgenetic haploid embryonic stem cell (AG-haESC) in which H19 DMR and IG-DMR are both knocked out with an oocyte to obtain a semi-cloned embryo, and incubating the semi-cloned embryo to obtain a semi-cloned mouse animal, wherein the AG-haESC is derived from a mouse; wherein one or more target genes of interest in the AG-haESC in which H19 DMR and IG-DMR are both knocked out are modified.