The present invention relates to methods for transfecting cells. In particular, the present invention relates to methods of transfecting primordial germ cells in avians, and to methods of breeding avians with modified traits.

The present invention relates to methods for transfecting cells. In particular, the present invention relates to methods of transfecting primordial germ cells in avians, and to methods of breeding avians with modified traits.

The present disclosure provides methods of generating multiplexed genetically modified animals, for example, porcine endogenous retrovirus (PERV)-inactivated pigs. The disclosure also provides methods of improving the birth rate of multiplexed genetically modified animals. In some embodiments, the present closure is concerned with the generation and utilization of porcine cells in which porcine endogenous retroviral (PERV) elements have been inactivated. In sonic embodiments, the PERV-free or PERV-reduced porcine cells are cloned to produce porcine embryos. In some embodiments, the PERV-free or PERV-reduced embryos may be grown into adult swine from which organs and/or tissues may be extracted and used for such purposes as xenotransplantation into non-porcine animals such as humans.

A method for producing a cell in which a target gene is knocked out, the method including the step of: introducing a CRISPR-Cas system into a cell having one or more kinds of target genes, the CRISPR-Cas system being able to produce (i) three or more kinds of guide RNAs for each of the one or more kinds of target genes and (ii) a Cas protein. The present invention can provide a method that enables highly efficient (90% or more) production of whole-body biallelic knockout animals in a single generation.

A method for producing a cell in which a target gene is knocked out, the method including the step of: introducing a CRISPR-Cas system into a cell having one or more kinds of target genes, the CRISPR-Cas system being able to produce (i) three or more kinds of guide RNAs for each of the one or more kinds of target genes and (ii) a Cas protein. The present invention can provide a method that enables highly efficient (90% or more) production of whole-body biallelic knockout animals in a single generation.

The present invention relates to the field of genome editing. More specifically, the present invention provides compositions and methods useful in clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques. In one embodiment, the present invention provides a double-stranded, linear donor polynucleotide comprising a template polynucleotide flanked by a first homology arm and a second homology arm, wherein the homology arms are between 30-35 bases in length.

The present invention relates to the field of genome editing. More specifically, the present invention provides compositions and methods useful in clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques. In one embodiment, the present invention provides a double-stranded, linear donor polynucleotide comprising a template polynucleotide flanked by a first homology arm and a second homology arm, wherein the homology arms are between 30-35 bases in length.

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.

The present disclosure provides a promoter having at least the core components of a duck retinoic acid-inducible gene I (RIG-I) promoter, as well as expression constructs having the duck RIG-I promoter operably linked to a gene product-encoding nucleic acid (e.g., an avian RIG-I protein), and recombinant host cells containing the duck RIG-I promoter, e.g., in such expression constructs. The present disclosure also provide animals genetically modified to have a gene encoding a duck RIG-I promoter operably linked to a gene product-encoding nucleic acid (e.g., an avian RIG-I protein, such as a duck RIG-I protein).

The present disclosure provides a promoter having at least the core components of a duck retinoic acid-inducible gene I (RIG-I) promoter, as well as expression constructs having the duck RIG-I promoter operably linked to a gene product-encoding nucleic acid (e.g., an avian RIG-I protein), and recombinant host cells containing the duck RIG-I promoter, e.g., in such expression constructs. The present disclosure also provide animals genetically modified to have a gene encoding a duck RIG-I promoter operably linked to a gene product-encoding nucleic acid (e.g., an avian RIG-I protein, such as a duck RIG-I protein).