Immunocompatible pluripotent stem cells (pSCs), which include cells compatible with different patient populations or patient-specific cells, find wide application in regenerative medicine therapies. Described herein are immunocompatible pSCs generated using techniques such as parthenogenesis resulting in cells possessing desired haplotypes of reduced zygosity, antigenically compatible with multiple patient populations, or nuclear transfer allowing generation of patient-specific cells. Methods described herein related to parthenogenesis, nuclear transfer, or pSC cell line generation. Also described herein are compositions of immunocompatible pSCs and cell lines generated by the aforementioned techniques.

The present invention relates to an AG-haESCs in which H19 DMR and IG-DMR are knocked out, a method for preparing the AG-haESCs, and use of the AG-haESCs in constructing a genetically modified semi-cloned animal and a library of a genetically modified semi-cloned animal. The AG-haESCs is capable of obtaining characteristics resembling a round spermatid, and upon injection into an oocyte, a viable SC mouse is stably obtained. The present invention is capable of being effectively used in multi-gene genetic manipulation, advancing the acquisition of animals with multiple genetic modifications.

The present invention relates to an AG-haESCs in which H19 DMR and IG-DMR are knocked out, a method for preparing the AG-haESCs, and use of the AG-haESCs in constructing a genetically modified semi-cloned animal and a library of a genetically modified semi-cloned animal. The AG-haESCs is capable of obtaining characteristics resembling a round spermatid, and upon injection into an oocyte, a viable SC mouse is stably obtained. The present invention is capable of being effectively used in multi-gene genetic manipulation, advancing the acquisition of animals with multiple genetic modifications.

Disclosed are methods and compositions for in situ germline genome engineering. The disclosed methods and compositions may be utilized for germline genome engineering in a subject having a reproductive organ containing a fertilized zygote, via: (i) isolating or obtaining the reproductive organ from the subject after a time period following insemination of the subject; (ii) introducing a reagent composition into the reproductive organ, the reagent composition comprising a nuclease system and/or an exogeneous polynucleotide; and (iii) electroporating the reproductive organ.

A method of constructing a zebrafish notch1a mutant using CRISPR/Cas9 technique. The method includes: determining a target for knocking out notch1a; using primers T7-notch1a-sfd and tracr rev for PCR amplification with a pUC19-gRNA scaffold plasmid as a template; transcribing PCR product in vitro followed by purification to obtain gRNA; and microinjecting the gRNA and a Cas9 mRNA into a zebrafish embryo followed by culture to obtain an notch1a mutant of stable inheritance. The invention selects a specific target and utilizes CRISPR/Cas9 technique to knock out the notch1a in the zebrafish without destroying other genes, generating the zebrafish notch1a mutant. Moreover, the invention also discloses the phenotype of the zebrafish notch1a mutant, which plays a significant role in studying the effect of the Notch1a receptor in the Notch signaling pathway.

A method of constructing a zebrafish notch1a mutant using CRISPR/Cas9 technique. The method includes: determining a target for knocking out notch1a; using primers T7-notch1a-sfd and tracr rev for PCR amplification with a pUC19-gRNA scaffold plasmid as a template; transcribing PCR product in vitro followed by purification to obtain gRNA; and microinjecting the gRNA and a Cas9 mRNA into a zebrafish embryo followed by culture to obtain an notch1a mutant of stable inheritance. The invention selects a specific target and utilizes CRISPR/Cas9 technique to knock out the notch1a in the zebrafish without destroying other genes, generating the zebrafish notch1a mutant. Moreover, the invention also discloses the phenotype of the zebrafish notch1a mutant, which plays a significant role in studying the effect of the Notch1a receptor in the Notch signaling pathway.

Semen and sperm cell processing and preservation systems, and methods of producing a mammal and methods of producing mammalian embryos are disclosed. The present invention is directed to sperm cell preservation, fertilization, and insemination, maintaining or enhancing sperm quality and addressing one or more sperm cell characteristics, such as viability, motility, functionality, fertilization rates, and pregnancy rates. Further, sperm cell characteristics may be addressed within the context of various collection, handling, separation, storage, transportation, usage, fertilization, or insemination techniques.

Semen and sperm cell processing and preservation systems, and methods of producing a mammal and methods of producing mammalian embryos are disclosed. The present invention is directed to sperm cell preservation, fertilization, and insemination, maintaining or enhancing sperm quality and addressing one or more sperm cell characteristics, such as viability, motility, functionality, fertilization rates, and pregnancy rates. Further, sperm cell characteristics may be addressed within the context of various collection, handling, separation, storage, transportation, usage, fertilization, or insemination techniques.

The present invention provides a genetic modification non-human organism in which an expression level of Cas9 is high and a plurality of different genes or a plurality of different locations in the same gene can be edited at the same time with high efficiency. The genetic modification non-human organism of the present invention includes a nuclear genome having at least 3 copies of genes that code for Cas9 (CRISPR-associated 9). Egg cells of the present invention are derived from the genetic modification non-human organism having the nuclear genome into which at least 3 copies of genes that code for the Cas9 are introduced. Fertilized eggs of the present invention are obtained by fertilizing the egg cells and sperm derived from the same species of the organism. A method for modifying target genes of the present invention includes a step of introducing a guide RNA into cells derived from the genetic modification non-human organism, the egg cells, or the fertilized eggs.

The present invention provides a genetic modification non-human organism in which an expression level of Cas9 is high and a plurality of different genes or a plurality of different locations in the same gene can be edited at the same time with high efficiency. The genetic modification non-human organism of the present invention includes a nuclear genome having at least 3 copies of genes that code for Cas9 (CRISPR-associated 9). Egg cells of the present invention are derived from the genetic modification non-human organism having the nuclear genome into which at least 3 copies of genes that code for the Cas9 are introduced. Fertilized eggs of the present invention are obtained by fertilizing the egg cells and sperm derived from the same species of the organism. A method for modifying target genes of the present invention includes a step of introducing a guide RNA into cells derived from the genetic modification non-human organism, the egg cells, or the fertilized eggs.