RESISTANT SWINE CROSS REFERENCE

[0001] This Application claims priority to U.S. Provisional Application No. 62/824,624, filed March 27, 2019, which is incorporated herein by reference in its entirety.

FIELD

[0002] The invention is directed to livestock animals, and in particular swine, that are resistant to the porcine reproductive and respiratory syndrome virus (PRRSV).

BACKGROUND

[0003] Porcine reproductive and respiratory syndrome virus (PRRSV) is a virus that causes a disease of pigs, called porcine reproductive and respiratory syndrome (PRRS). PRRS is a panzootic disease that causes reproductive failure and respiratory illness in immature pigs. PRRSV is an economically important disease with costing an estimated $644 million a year in the United States and 1.5b€ annually.

SUMMARY

[0004] Disclosed herein is a method of producing a genetically modified livestock cell, the method comprising the steps of introducing into the livestock cell a nuclease with a guide RNA, wherein the guide RNA binds to a target sequence in SEQ ID NO. 87, wherein the target sequence is between intron 6 and exon 7 of the CD163 gene; and editing the target sequence of the livestock cell genome such that position 12 of SEQ ID NO: 87 is modified from an adenine to an inosine. The livestock cell can be a porcine cell. The livestock cell can be a porcine fibroblast. The guide RNA can be configured to target SEQ ID NO: 88. The nuclease can be a base editor. The editor can be an adenine base editor. Introducing can comprise transfecting the livestock cell with RNA encoding a codon optimized base editor. The base editor can be an adenine base editor. Position 12 of SEQ ID NO: 87 can be modified from an inosine to a guanine after replication of the genome. In some embodiments, the editing does not comprise a repair template. Introducing can comprise transfecting the livestock cell with a base editor protein to form a base editor ribonuclease complex. The base editor can be an adenine base editor.

[0005] Disclosed herein is a genetically modified livestock animal wherein a genome of the genetically modified livestock animal is modified by a nuclease complex, wherein the nuclease complex comprises a nuclease and a guide RNA, wherein the modification of the genome is such that position 12 of SEQ ID NO: 87 of the livestock animal genome is modified from an A to one of G, T, or C, wherein a repair template is not used. The genetic modification can render the livestock animal resistant to porcine reproductive and respiratory syndrome virus (PRRSV).

[0006] Disclosed herein is a method of producing an animal resistant to porcine reproductive and respiratory syndrome virus (PRRSV), the method comprising introducing into a cell isolated from the animal a base editor, wherein the base editor modifies the CD163 gene in the cell. The base editor can comprise a nuclease and a guide RNA. The guide RNA can target SEQ ID NO.: 87. The animal can be porcine. The cell can be a porcine cell. The cell can be a porcine fibroblast. The guide RNA can be configured to target SEQ ID NO: 88. The base editor can be an adenine base editor. Position 12 of SEQ ID NO: 87 can be modified from an adenine to an inosine. Position 12 of SEQ ID NO.87 can be modified from an inosine to a guanine after replication of the genome. In some instances, modifying does not comprise a repair template.

[0007] Disclosed herein is a method of producing a livestock animal cell that expresses a modified CD163 protein that is resistant to porcine reproductive and respiratory syndrome virus (PRRSV), the method comprising substituting or deleting one or more bases of the genome of the livestock animal cell within one or more codons corresponding to one or more of amino acids of a scavenger receptor cysteine-rich domain 5 (SRCR5) of a native CD163 protein using a nuclease targeting the native CD163 gene sequence of the native CD163 protein, wherein the one or more amino acids are selected from the group consisting of Q488, T495, L521, S531, and D558 of SEQ ID NO: 59. The SRCR5 domain of the modified CD163 protein can comprise the following amino acid sequence:

PRLVGGDIPCSGVEVx1HGDTWGx2VCDSDFSLEAASVLCELQCGTVVSx3LGGAHF GEG x4GQIWAEEFQCEGFESGLSLCPVAPRPx5GTCSHSRDVGVVCS, wherein each of x1, x2, x3, x4, and x5 are selected from the group consisting of G, K, S, I, N or E. The deleting further can comprise deleting bases in discrete regions of a gene sequence corresponding to the SRCR5 of the native CD163 protein. The method can further comprise an in-frame deletion of a residue unique to swine. The method can further comprise a deletion ranging in size from 66 bases to 210 bases, or any combination thereof.

[0008] Disclosed herein is a genetically edited porcine animal that expresses a modified CD163 protein wherein a CD163 gene sequence of a genome of the genetically modified porcine animal comprises a modification as compared to a wild type CD163 gene sequence, to one or more codons coding for one or more of amino acids of the modified CD163 protein, wherein the one or more amino acids are selected from the group consisting of: Q488, T495, L521, S531, or D558 of the wild type CD163 protein. The genetic modification can be an in-frame deletion. The genetic modifications can comprise one or more amino acid conversions comprising Q488K, T495S, L521I, S531N or D558E. The genetic modifications can consist of the amino acid conversions Q488K, T495S, L521I, S531N and D558E. The genetic modification can eliminate the ability of CD163 to bind to a porcine reproductive and respiratory syndrome virus (PRRSV).

[0009] These and other features and advantages of the present invention will be set forth or will become more fully apparent in the description that follows and in the appended claims. The features and advantages may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. Furthermore, the features and advantages of the invention may be learned by the practice of the invention or will be apparent from the description, as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various exemplary embodiments of the compositions and methods according to the invention will be described in detail, with reference to the following figures wherein:

[0011] FIG. 1 is a cartoon of the CD163 gene. There are 16 exons and 9 SRCR domains with exon 16 being the sole intracellular domain.

[0012] FIG.2: is an alignment of exon 7/SRCR5 of CD163 of swine (ss), human (hs), mouse (mm) and monkey (ca).

[0013] FIG.3: is the amino acid sequence of the CD163 protein with exon 7 and SRCR5 identified;

[0014] FIG.4: illustrates one strategy of editing the CD163 gene by deleting E537-V558 (22 aa: EEFQCEGHES HLSLCPVAPRPD) including the unique swine aa D5558 using TALENs or CRISPR/CAS9 using an ssODN template.

[0015] FIG.5: illustrates a second strategy of making the same edit as illustrated in FIG.4, with the exception that a short homology arm dsODN template is used.

[0016] FIG.6: shows another strategy for editing the CD163 gene by deleting E537-V567 (31 aa: EEFQCEGHESHLSLCPVAPRPDGTCSHSRDV) using an ssODN template and either TALENs or CRISPR/CAS9.

[0017] FIG.7 illustrates a separate strategy for making the same deletion as shown in FIG.6 with the exception that a short homology arm dsODN template is used.

[0018] FIG.8 illustrates yet a different strategy for editing the CD163 gene by deletion of S520- C571 (52aa: SLGAHFGEGSGQIWAEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVC) including three unique swine amino acids using an ssODN template and either TALENs or CRISPR/CAS9.

[0019] FIG.9 illustrates a separate strategy for making the same deletion as shown in FIG.8 with the exception that a short homology arm dsODN template is used. [0020] FIG. 10 illustrates a different strategy for editing the CD 163 gene to delete S482-V496 (15 aa: SGRVEVQHGDTWGTV) including two unique swine amino acids using a ssODN template and either TALENs or CRISPR/CAS9.

[0021] FIG. 11 illustrates a separate strategy for making the same deletion as shown in FIG. 10 with the exception that a short homology arm dsODN template is used.

[0022] FIG. 12 illustrates a different strategy for editing the CD163 gene to delete Q488-S531 (44aa: QHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFGEGS) including four unique swine amino acids using a ssODN template and either TALENs or CRISPR/CAS9;

[0023] FIG. 13 illustrates a separate strategy for making the same deletion as shown in FIG. 12 with the exception that a short homology arm dsODN template is used.

[0024] FIG. 14A-14E illustrate a modification by an adenine base editor (ABE) to the splice acceptor site between intron 6 and exon 7 of the CD163 gene which prevents the translation of exon 7 of the CD163 gene, which codes for the SRCR5 trans membrane domain of the CD163 protein.

[0025] FIG.15A-15D illustrate a modification by a CRISPR Cas9 nuclease and a repair template integrated by homology directed repair (HDR) which modifies the splice acceptor site between intron 6 and exon 7 of the CD163 gene which prevents the translation of exon 7 of the CD163 gene, which codes for the SCRC5 trans membrane domain of the CD163 protein.

[0026] FIG.16A-16C illustrate a modification by a prime editor to the splice acceptor site between intron 6 and exon 7 of the CD163 gene which prevents the translation of exon 7 of the CD163 gene, which codes for the SRCR5 trans membrane domain of the CD163 protein.

DETAILED DESCRIPTION

[0027] PRRSV is highly contagious and is an enveloped RNA virus. It contains a single-stranded, positive-sense, RNA genome with a size of approximately 15 kilobases in a genome that contains nine open reading frames. There are two main strains of PRRSV, the“North American” strain (VR-2332) and the European strain (the“Lelystad virus” (LV)). A highly pathogenic (HP) strain of the North American genotype emerged in China in the early 2000s. The HP-PRRSV, is more virulent and has caused significant losses throughout Asia.

[0028] The PRRS virus has been shown to infect cells of the monocyte/macrophage family via two specific surface receptors CD163 and CD169. Macrophages, of particular subtypes express, CD163. Pigs in which the CD163 gene has been knocked-out have been shown to be resistant to PRRSV infection.

[0029] CD163 has been identified, in pigs, as comprising a gene of approximately 104,526,007 bp (NC_010447) having 16 exons coding for an approximately 3,400bp mRNA (NM_213976) sequence that codes for a 1110 aa receptor (NM_213976) having a single short transmembrane domain and a short intracellular domain. Exons 2-13 are predicted to encode nine scavenger receptor cysteine-rich (SRCR) domains with SRCR 9 being connected to a transmembrane domain and a short cytoplasmic domain via a proline-serine-threonine (PST)-RICH GEGION. The CD163 receptor is found on cells of the monocyte/macrophage lineage. It has been found that a knockout of the CD163 gene results in an animal that is resistant to PRRSV infection. However, the CD163 protein appears to play an important role in macrophage function, the immune response and the removal of hemoglobin from the blood. Therefore, a complete knockout of this gene may not result in pigs that are viable or fit. The transmembrane domain and SRCR5 have been found to be essential for the infection by PRRSV with SRCR5 being found entirely within exon 7, (FIG.1).

[0030] Previous groups have made various modifications to the CD163 gene in an effort to provide animals that are resistant to PRRSV. These modifications include a complete knockout of the gene and various deletions of regions within the gene, which result in a knockout of the protein and a complete deletion of exon 7 which is facilitated by the fact that this deletion is in-frame and therefore a functional protein is thought to be expressed by the truncated gene. Because of its highly conserved nature and its normal physiological role in immune activities, it would be desirable to provide an animal with a largely intact CD163 protein that does not contribute to PRRSV infectivity.

[0031] Swine that are resistant to or not prone to infection by the porcine reproductive and respiratory syndrome virus (PRRSV) are provided. The animals are gene edited to delete discrete portions of the swine CD163 gene or to convert those amino acid residues in the CD163 protein which are not conserved (see, for example FIG. 2) and which are required for infectivity and virulence.

[0032] Thus, in one exemplary embodiment, the disclosure provides a genetically edited porcine animal comprising a genetic modification which alters the expression or activity of CD 163, and wherein the protein retains a majority of exon 7. In these and other embodiments, the modification comprises in-frame deletions of portions exon 7 (and SRCR5). In some embodiments, the in- frame deletions result in the deletion of amino acids from the protein that are unique to the swine. In various exemplary embodiments, the porcine animal has been edited to eliminate the ability of porcine reproductive and respiratory syndrome virus (PRRSV) to bind to CD163. In these and other embodiments, the edits to the CD163 gene comprise deletions of Q488, T495, L521, S531, D558 and combinations thereof of the CD163 protein. In some embodiments, the deletions range in size from 1 aa to 52 aa. In these embodiments, the deletions range in size from 3 to 6 to 156 basepairs (bp). [0033] In yet other exemplary embodiments, the CD163 gene is edited to convert amino acids unique in the swine protein to amino acids conserved throughout non-susceptible species. In these embodiments, the conversions comprise Q488K, T495S, L521I, S531N and D558E.

[0034] In yet other embodiments, the CD163 gene is edited to change or inactivate the intron 6 splice acceptor. In these embodiments, the splice acceptor can be edited to any nonconsensus “AG” sequence. In various exemplary embodiments the exon 6 splice acceptor is edited with a base editor to convert“AG” to“GG”.

[0035] In yet other embodiments the disclosure provides a method to provide porcine animals that are resistant to PRRSV comprising: editing the CD163 gene of the animal to delete or convert Q488, T495, L521, S531, D558 and combinations thereof. In these and other embodiments the methods provide swine having edits leaving a majority of the CD163 gene intact or converting the unique swine amino acids to different amino acids. In various exemplary embodiments, Q488, T495, L521, S531, D558 are converted to K, S, I, N and E respectively. In other exemplary embodiments, deletions are made in the SRCR5 domain of ranging in size from 1 aa to 52 aa. In these embodiments, the edits to the gene range in size from 3 to 6 bp.

[0036] Previous groups have made various modifications to the CD163 gene in an effort to provide animals that are resistant to PRRSV. These modifications include inactivation of the gene via out- of-frame deletions and insertions and replacement of SRCR5 with a human homolog of the SRCR8 domain and a complete deletion of exon 7 which is facilitated by the fact that this deletion is in- frame and therefore a functional protein is expressed by the gene deletion. As disclosed herein, provided is a porcine animal with discrete deletions or conversions of amino acids unique to the swine SRCR5 which results in SRCR functionality and PRRSV resistance.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the disclosure. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0038] It must be noted that as used herein and in the appended claims, the singular forms“a”, “an”, and“the” include plural reference unless the context clearly dictates otherwise. As well, the terms“a” (or“an”),“one or more” and“at least one” can be used interchangeably herein. It is also to be noted that the terms“comprising”,“including”,“characterized by” and“having” can be used interchangeably.

[0039]“Additive Genetic Effects” as used herein means average individual gene effects that can be transmitted from parent to progeny.

[0040]“Allele” as used herein refers to an alternate form of a gene. It also can be thought of as variations of DNA sequence. For instance, if an animal has the genotype for a specific gene of Bb, then both B and b are alleles.

[0041] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0042] The term“and/or” means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase“one or more” is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0043] As used herein,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating a listing of items,“and/or” or“or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of or“exactly one of,” or, when used in the claims,“consisting of” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only he interpreted as indicating exclusive alternatives (i.e.,“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”

[0044] As used herein, the terms“including”,“includes”,“having”,“has”,� ��with”, or variants thereof, are intended to be inclusive similar to the term“comprising.”

[0045]“DNA Marker” refers to a specific DNA variation that can be tested for association with a physical characteristic. [0046] The term“about” can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example,“about 50” percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term“about” can include one or two integers greater than and/ or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term“about” is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the end-points of a recited range.

[0047] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term “about.” These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0048] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as“up to,”“at least,”“greater than,”“less than,”“more than,”“or more,” and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0049]“Genome” refers to the genetic makeup of an animal that is the total complement of DNA in its chromosomes.

[0050]“Genotype” refers to a particular sequence and a particular allele or loci

[0051]“Genotyping (DNA marker testing)” refers to the process by which an animal is tested to determine the particular alleles it is carrying for a specific genetic test. Organisms may be genotyped to identify various genetic markers. Genetic markers can be a sequence comprising a plurality of bases, or a single nucleotide polymorphism (SNP) at a known location.

[0052]“Simple Traits” refers to traits such as coat color and horned status and some diseases that are carried by a single gene.

[0053]“Complex Traits” refers to traits such as reproduction, growth and carcass that are controlled by numerous genes.

[0054]“Complex allele”–coding region that has more than one mutation within it. This makes it more difficult to determine the effect of a given mutation because researchers cannot be sure which mutation within the allele is causing the effect.

[0055]“Copy number variation” (CNVs) a form of structural variation - are alterations of the DNA of a genome that results in the cell having an abnormal or, for certain genes, a normal variation in the number of copies of one or more sections of the DNA. CNVs correspond to relatively large regions of the genome that have been deleted (fewer than the normal number) or duplicated (more than the normal number) on certain chromosomes. For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-“Repetitive element” patterns of nucleic acids (DNA or RNA) that occur in multiple copies throughout the genome. Repetitive DNA was first detected because of its rapid association kinetics.

[0056]“Quantitative variation” variation measured on a continuum (e.g. height in human beings) rather than in discrete units or categories. See continuous variation. The existence of a range of phenotypes for a specific character, differing by degree rather than by distinct qualitative differences.

[0057]“Homozygous” refers to having two copies of the same allele for a single gene such as BB.

[0058]“Heterozygous” refers to having different copies of alleles for a single gene such as Bb.”

[0059]“Locus” (plural“loci”) refers to the specific locations of a maker or a gene.

[0060]“Centimorgan (Cm)” a unit of recombinant frequency for measuring genetic linkage. It is defined as the distance between chromosome positions (also termed, loci or markers) for which the expected average number of intervening chromosomal crossovers in a single generation is 0.01. It is often used to infer distance along a chromosome. It is not a true physical distance however.

[0061]“Chromosomal crossover” (“crossing over”) is the exchange of genetic material between homologous chromosomes inherited by an individual from its mother and father. Each individual has a diploid set (two homologous chromosomes, e.g., 2n) one each inherited from its mother and father. During meiosis I the chromosomes duplicate (4n) and crossover between homologous regions of chromosomes received from the mother and father may occur resulting in new sets of genetic information within each chromosome. Meiosis I is followed by two phases of cell division resulting in four haploid (1n) gametes each carrying a unique set of genetic information. Because genetic recombination results in new gene sequences or combinations of genes, diversity is increased. Crossover usually occurs when homologous regions on homologous chromosomes break and then reconnect to the other chromosome.

[0062]“Marker Assisted Selection (MAS)” refers to the process by which DNA marker information is used to assist in making management decisions.

[0063]“Marker Panel” a combination of two or more DNA markers that are associated with a particular trait.

[0064]“Non-additive Genetic Effects” refers to effects such as dominance and epistasis. Codominance is the interaction of alleles at the same locus while epistasis is the interaction of alleles at different loci.

[0065]“Nucleotide” refers to a structural component of DNA that includes one of the four base chemicals: adenine (A), thymine (T), guanine (G), and cytosine (C).

[0066]“Phenotype” refers to the outward appearance of an animal that can be measured. Phenotypes are influenced by the genetic makeup of an animal and the environment.

[0067]“Single Nucleotide Polymorphism (SNP)” is a single nucleotide change in a DNA sequence.

[0068]“Haploid genotype” or“haplotype” refers to a combination of alleles, loci or DNA polymorphisms that are linked so as to cosegregate in a significant proportion of gametes during meiosis. The alleles of a haplotype may be in linkage disequilibrium (LD).

[0069]“Linkage disequilibrium (LD)” is the non-random association of alleles at different loci i.e. the presence of statistical associations between alleles at different loci that are different from what would be expected if alleles were independently, randomly sampled based on their individual allele frequencies. If there is no linkage disequilibrium between alleles at different loci they are said to be in linkage equilibrium.

[0070] The term“restriction fragment length polymorphism” or“RFLP” refers to any one of different DNA fragment lengths produced by restriction digestion of genomic DNA or cDNA with one or more endonuclease enzymes, wherein the fragment length varies between individuals in a population.

[0071]“Introgression” also known as“introgressive hybridization”, is the movement of a gene or allele (gene flow) from one species into the gene pool of another by the repeated backcrossing of an interspecific hybrid with one of its parent species. Purposeful introgression is a long-term process; it may take many hybrid generations before the backcrossing occurs. [0072]“Nonmeiotic introgression” genetic introgression via introduction of a gene or allele in a diploid (non-gametic) cell. Non-meiotic introgression does not rely on sexual reproduction and does not require backcrossing and, significantly, is carried out in a single generation. In non- meiotic introgression an allele is introduced into a haplotype via homologous recombination. The allele may be introduced at the site of an existing allele to be edited from the genome or the allele can be introduced at any other desirable site.

[0073] As used herein the term“genetic modification” refers to is the direct manipulation of an organism's genome using biotechnology.

[0074] As used herein the phrase“precision gene editing” means a process gene modification which allows geneticists to introduce (introgress) any natural trait into any breed, in a site-specific manner without the use of recombinant DNA.

[0075] As used herein, the term“gene conversion” means editing a gene to comprise an insertion, a deletion, the insertion of an exogenous nucleic acid fragment, an inversion, a gene conversion to natural allele, gene conversion to a synthetic allele, and a gene conversion to a novel allele. The term natural allele in the context of genetic modification means an allele found in nature in the same species of organism that is being modified. The term novel allele means a non-natural allele. A human allele placed into a goat is a novel allele. The term synthetic allele means an allele that is not found in nature. Thus, a natural allele is a variation already existing within a species that can be interbred. And a novel allele is one that does not exist within a species that can be interbred.

[0076]“Transcription activator-like effector nucleases (TALENs)” one technology for gene editing are artificial restriction enzymes generated by fusing a TAL effector DNA-binding domain to a DNA cleavage domain.

[0077]“Zinc finger nucleases (ZFNs)” as used herein are another technology useful for gene editing and are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.

[0078]“Meganuclease” as used herein are another technology useful for gene editing and are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result, this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-SceI meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome). Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes. [0079]“CRISPR/CAS” technology as used herein refers to“CRISPRs” (clustered regularly interspaced short palindromic repeats), segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of“spacer DNA” from previous exposures to a bacterial virus or plasmid.“CAS” (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location.

[0080]“Indel” as used herein is shorthand for“insertion” or“deletion” referring to a modification of the DNA in an organism.

[0081] As used herein the term“renucleated egg” refers to an enucleated egg used for somatic cell nuclear transfer in which the modified nucleus of a somatic cell has been introduced.

[0082]“Genetic marker” as used herein refers to a gene/allele or known DNA sequence with a known location on a chromosome. The markers may be any genetic marker e.g., one or more alleles, haplotypes, haplogroups, loci, quantitative trait loci, or DNA polymorphisms [restriction fragment length polymorphisms(RFLPs), amplified fragment length polymorphisms (AFLPs), single nuclear polymorphisms (SNPs), indels, short tandem repeats (STRs), microsatellites and minisatellites]. Conveniently, the markers are SNPs or STRs such as microsatellites, and more preferably SNPs. Preferably, the markers within each chromosome segment are in linkage disequilibrium.

[0083] As used herein the term“host animal” means an animal which has a native genetic complement of a recognized species or breed of animal.

[0084] As used herein,“native haplotype” or“native genome” means the natural DNA of a particular species or breed of animal that is chosen to be the recipient of a gene or allele that is not present in the host animal.

[0085] As used herein the term“target locus” means a specific location of a known allele on a chromosome.

[0086] As used herein, the term“quantitative trait” refers to a trait that fits into discrete categories. Quantitative traits occur as a continuous range of variation such as that amount of milk a particular breed can give or the length of a tail. Generally, a larger group of genes controls quantitative traits.

[0087] As used herein, the term“qualitative trait” is used to refer to a trait that falls into different categories. These categories do not have any certain order. As a general rule, qualitative traits are monogenic, meaning the trait is influenced by a single gene. Examples of qualitative traits include blood type and flower color, for example.

[0088] As used herein, the term“quantitative trait locus (QTL)” is a section of DNA (the locus) that correlates with variation in a phenotype (the quantitative trait). [0089] As used herein the term“cloning” means production of genetically identical organisms asexually.

[0090]“Somatic cell nuclear transfer” (“SCNT”) is one strategy for cloning a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte (egg cell) and implanting a donor nucleus from a somatic (body) cell.

[0091]“Orthologous” as used herein refers to a gene with similar function to a gene in an evolutionarily related species. The identification of orthologues is useful for gene function prediction. In the case of livestock, orthologous genes are found throughout the animal kingdom and those found in other mammals may be particularly useful for transgenic replacement. This is particularly true for animals of the same species, breed or lineages wherein species are defined two animals so closely related as to being able to produce fertile offspring via sexual reproduction; breed is defined as a specific group of domestic animals having homogenous phenotype, homogenous behavior and other characteristics that define the animal from others of the same species; and wherein lineage is defined as continuous line of descent; a series of organisms, populations, cells, or genes connected by ancestor/descendent relationships. For example, domesticated cattle are of two distinct lineages both arising from ancient aurochs. One lineage descends from the domestication of aurochs in the Middle East while the second distinct lineage descends from the domestication of the aurochs on the Indian subcontinent.

[0092]“Genotyping” or“genetic testing” generally refers to detecting one or more markers of interest e.g., SNPs in a sample from an individual being tested, and analyzing the results obtained to determine the haplotype of the subject. As will be apparent from the disclosure herein, it is one exemplary embodiment to detect the one or more markers of interest using a high-throughput system comprising a solid support consisting essentially of or having nucleic acids of different sequence bound directly or indirectly thereto, wherein each nucleic acid of different sequence comprises a polymorphic genetic marker derived from an ancestor or founder that is representative of the current population and, more preferably wherein said high-throughput system comprises sufficient markers to be representative of the genome of the current population. Preferred samples for genotyping comprise nucleic acid, e.g., RNA or genomic DNA and preferably genomic DNA. A breed of livestock animal can be readily established by evaluating its genetic markers.

[0093] The term“proximate” as used herein means close to.

[0094] Splice Acceptor Introns have two distinct nucleotides at either end. At the 5’ end of the intron, the DNA nucleotides are GT (the splice donor) and at the 3’ end they are AG (the splice acceptor). The splice acceptor and the splice donor are recognized by the spliceosome for of the intervening sequences, the introns, when chromosomal DNA it is transcribed into mRNA (pre- mRNA).

[0095]“Base Editing”: Base editing is a form of genome editing that enables direct, irreversible conversion of one base pair to another at a target genomic locus without requiring double-stranded DNA breaks (DSBs), homology-directed repair (HDR) processes, or donor DNA templates. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme, and in some cases a glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts.

[0096]“Prime Editing”: Prime editing is a form of precise genome editing that enables direct, irreversible targeted small insertions, deletions, and base swapping without requiring double- stranded DNA breaks (DSBs), or donor DNA templates. DNA prime editors comprise a catalytically disabled Cas9 nuclease fused to a reverse transcriptase. Prime editing involves a prime editing guide RNA (pegRNA) that is substantially larger than a standard sgRNA used for CRISPR Cas9 editing. The pegRNA comprises a primer binding sequence (PBS) and a template containing the desired RNA sequence added at the 3’ end.

[0097] Generation of a double strand break does not directly lead to DNA editing. Editing following nuclease treatment occurs as a result of cellular responses to the double strand break. Processes include non-homologous end joining (NHEJ) and microhomology-mediated end-joining (MMEJ) which can lead to gene disruption through the introduction of insertions, deletions, translocations, or other DNA rearrangements at the site of a double strand break. Alternatively, a precise DNA edit can be made by supplying a donor DNA template encoding the desired DNA change flanked by sequence homologous to the region upstream and downstream of the double strand break. Cellular homology direct repair (HDR) then results in the incorporation of sequence from the exogenous DNA template at the double strand break site.

[0098] Homology directed repair (HDR) is a mechanism in cells to repair ssDNA and double stranded DNA (dsDNA) lesions. This repair mechanism can be used by the cell when there is an HDR template present that has a sequence with significant homology to the lesion site. Specific binding, as that term is commonly used in the biological arts, refers to a molecule that binds to a target with a relatively high affinity compared to non-target tissues, and generally involves a plurality of non-covalent interactions, such as electrostatic interactions, van der Waals interactions, hydrogen bonding, and the like. Specific hybridization is a form of specific binding between nucleic acids that have complementary sequences. Proteins can also specifically bind to DNA, for instance, in TALENs or CRISPR/Cas9 systems or by Gal4 motifs. Introgression of an allele refers to a process of copying an exogenous allele over an endogenous allele with a template-guided process. The endogenous allele might actually be excised and replaced by an exogenous nucleic acid allele in some situations, but present theory is that the process is a copying mechanism. Since alleles are gene pairs, there is significant homology between them. The allele might be a gene that encodes a protein, or it could have other functions such as encoding a bioactive RNA chain or providing a site for receiving a regulatory protein or RNA.

[0099] The HDR template is a nucleic acid that comprises a portion of an allele that is being introgressed, an exogenous sequence introduced into the genome or deletion of a portion of an allele. The template may be a dsDNA or a single-stranded DNA (ssDNA). ssDNA templates are preferably from about 20 to about 5000 residues although other lengths can be used. Artisans will immediately appreciate that all ranges and values within the explicitly stated range are contemplated; e.g., from 500 to 1500 residues, from 20 to 100 residues, and so forth. The template may further comprise flanking sequences that provide homology to DNA adjacent to the endogenous allele or the DNA that is to be replaced. Such flanking residues are termed“homology arms” and comprise from 5 to 10 to 40 and up to 200 and 500 bp or more on either side (e.g.,“left” and“right”“homology arms”) of the introgressed sequence. Artisans will immediately appreciate that all ranges and values within the explicitly stated range are contemplated. In those cases where a simple deletion is made, the HDR template may simply comprise a homologous sequence reading on either side of the deletion sequence. The template may also comprise a sequence that is bound to a targeted nuclease system and is thus the cognate binding site for the system’s DNA-binding member. The term cognate refers to two biomolecules that typically interact, for example, a receptor and its ligand. In the context of HDR processes, one of the biomolecules may be designed with a sequence to bind with an intended, i.e., cognate, DNA site or protein site.

[0100] Although HDR is a flexible tool with the ability to make precise insertions, deletions, or any point mutation of interest, HDR is largely restricted to the G2 and S phase of the cell cycle, limiting efficient HDR to actively dividing cells, and even in cultured cell lines HDR efficiency can be modest. Moreover, NHEJ and HDR are competing processes, and under most conditions NHEJ is more efficient than HDR. Thus, small insertions and deletions may appear in edited products. Thus, the use of a base editor or a prime editor, which do not directly generate double strand breaks, do not require a DNA donor template and do not rely on cellular HDR. Targeted Endonuclease Systems

[0101] Genome editing tools such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have impacted the fields of biotechnology, gene therapy and functional genomic studies in many organisms. More recently, RNA-guided endonucleases (RGENs) are directed to their target sites by a complementary RNA molecule. The Cas9/CRISPR system is an RGEN. Base editors are RGENs. Prime editors are RGENs. These are examples of targeted nuclease systems: these systems have a DNA-binding member that localizes the nuclease to a target site. The site is then modified by the nuclease. TALENs and ZFNs have the nuclease fused to the DNA-binding member. Cas9/CRISPR are cognates that find each other on the target DNA. Base editors comprise fusions between a catalytically impaired Cas nuclease and a base- modification enzyme that operates on single stranded DNA, but not double-stranded DNA. Prime editors comprise fusions between a catalytically impaired Cas nuclease and a reverse transcriptase that operates on single stranded DNA, but not double-stranded DNA. The DNA-binding member has a cognate sequence in the chromosomal DNA. The DNA-binding member is typically designed in light of the intended cognate sequence so as to obtain a nucleolytic action at or near an intended site. Certain embodiments are applicable to all such systems without limitation; including, embodiments that minimize nuclease re-cleavage, embodiments for making SNPs with precision at an intended residue, and placement of the allele that is being introgressed at the DNA- binding site. Base Editors

[0102] The term base editor, as used herein, is broad and includes a fusion protein comprising a catalytically impaired Cas nuclease and a base-modification enzyme that operates on single stranded DNA (ssDNA) or RNA. Upon binding to a target DNA locus, base pairing between the guide RNA and the target DNA strand lead to displacement of a small segment of single-stranded DNA in an“R-loop”. DNA bases within this single-stranded DNA R-loop are modified by the deaminase enzyme. The catalytically disabled Cas nuclease generates a nick in the non-edited DNA strand, inducing cells to repair the non-edited strand using the edited strand as a template.

[0103] Two classes of DNA base editor have been described: cytosine base editors (CBEs) that convert a C-G base pair to a T-A base pair, and adenine base editors (ABEs) that convert an A-T base pair to a G-C base pair. In RNA, targeted adenosine conversion to inosine has also been developed using both antisense and Cas13 guided RNA targeting methods.

[0104] CBEs convert a C-G base pair to a T-A base pair by deaminating the exocyclic amine of the target cytosine to generate uracil. To localize deamination activity to a small target window within a mammalian genome, a APOBEC1 cytidine deaminase can be used. APOBEC1 cytidine deaminase accepts single stranded DNA as a substrate and is incapable of acting on double stranded DNA. A Cas9 nuclease can be modified with a D10A and H840A substitution to produce a dCas9 with inhibited catalytic activity. A CBE can comprise an APOBEC1 fused to a dCas9. When bound to a target DNA, the dCas9 performs local denaturation of he DNA duplex to generate an R-loop in which the DNA strand not paired with the guide RNA exists as a disordered single- stranded bubble. This feature enables the CBE to perform efficient and localized cytosine deamination to a uridine, which is then base paired to an adenine. Upon replication, the uridine is replaced by a Thymine.

[0105] ABEs convert an A-T base pair to a G-C base pair by deamination of the adenosine to yield an inosine. Inosine can exhibit a base-pairing preference for guanosine. A tRNA adenosine deaminase enzyme, TadA, is a deoxyadenosine deaminase enzyme that accepts single stranded DNA and converts a deoxyadenosine to a deoxyinosine. It is known that a TadA-TadA*-Cas9 heterodimeric protein is highly efficient at localized deoxyadenosine deamination, wherein TadA is a wild-type non-catalytic TadA monomer and TadA* is an evolved TadA* monomer as described by Liu et al. (See Liu et al., Nature 2017 Nov.23; 551(7681):464-471).

[0106] Splicing out of introns during transcription is facilitated by the presence of a splice donor at the start of an intron (5’) and a splice acceptor at the end of the intron (3’). The splice donor has the consensus GT while the splice acceptor has the consensus AG. Small nuclear ribonucleoproteins (snRP’s) search for the splice acceptor and slice donor during transcription and join together the exons to provide the intron-less mRNA. If the splice donor or splice acceptor is mutated so as not to comprise the consensus, either the beginning of the intron or the end of the intron is not recognized. In this example, the splice acceptor is mutated so as not to comprise AG.

[0107] By changing the splice acceptor, the beginning of the intron will be recognized but the snRPs will not recognize the end of the intron and will instead pass on to the next splice acceptor. In this case, the next splice acceptor will be that at the end of intron 7 (just prior to exon 8). The result will be that the entire exon 7 will be spliced out of the mRNA with exon 6 being ligated directly to exon 8, thus also leaving the entire sequence of exon 7 intact within the chromosome. For this embodiment, base editing can be used. Specifically, the adenine (A) residue can be converted to inosine, by using an adenine base editor (ABE) as described herein. The nick caused by the ABE in the second strand will lead to repair by the cell to base pair the inosine with a cytosine (C). This base change will render the intron 6 splice acceptor unreadable resulting in the small nuclear ribonucleoproteins (snRPs) recognizing the 7 ^th intron splice acceptor as the pair of the 6 ^th intron splice donor and resulting in the excision of the 6 ^th intron, 7 ^th exon and 7 ^th intron during mRNA transcription. Upon replication of the genome, the inosine will be read as a guanine (G). The following gRNAs can be utilized to make the base changes: ssCD163 g7.8, ssCD163 g7.9 and ssCD163 g7.10.

[0108] ssCD163 g7.8

[0109] TTTCAGCCCACAGGAAACCC (SEQ ID NO: 44)

[0110] ssCD163 g7.9

[0111] GGGCTGAAAAAATAGCATTT (SEQ ID NO: 45)

[0112] ssCD163 g7.10

[0113] TTTCAGCCCACAGGAAACCC (SEQ ID NO: 88)

Prime Editors

[0114] Similar to CRISPR, prime editing requires the presence of a catalytically modified Cas endonuclease and a single guide RNA. The Cas9 endonuclease is catalytically modified to be a Cas9 nickase which nicks the DNA rather than generating a double-strand break. The Cas9 nickase is fused to a reverse transcriptase. The prime editing guide RNA (pegRNA), is substantially larger than standard sgRNA. The pegRNA is a sgRNA with a primer binding sequence (PBS) and the template containing the desired RNA sequence added at the 3’end.

[0115] As can be seen in FIG.16A, using the PBS, the pegRNA binds to the target site. The Cas9 nickase then creates a cut in the opposite single strand, and the desired RNA sequence binds to the cut single strand. The reverse transcriptase then reverse transcribes the RNA sequence and the new sequence is incorporated into the new DNA at the target site. This leaves one strand edited and one strand unedited. The edited strand is incorporated by flap equilibration. The original DNA segment is then removed by a cellular endonuclease. A second prime editor complexed with a standard guide RNA is then used to nick the unedited strand. The edited strand is then used as a template to repair the nick, thus completing the edit on both strands.

[0116] The following pegRNAs can be utilized to make the base changes: ssCD163 ex7 SA pegRNA-1 and ssCD163 ex7 SA pegRNA-2.

[0117] ssCD163 ex7 SA pegRNA-1

[0118] 5’ - AACCAGCCUGGGUUUCCUGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUG…GCACCGAGUCGGUGCCUAUUUUUUC XXCCCACAGGAAACCCAGGCUUUU - 3’ (SEQ ID NO: 92)

[0119] Bold: Spacer Binding Sequence

[0120] Italic: Reverse Transcriptase Template

[0121] Underline: Primer binding site [0122] Bold Italic: dinucleotide mutation to block exon 7 splicing; XX can be GG or AA.

[0123] ssCD163 ex7 SA pegRNA-2

[0124] 5’ - AUGUCCCCUCCAACCAGCCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG GCUAGUCCGUUAUCAACUUGAAAAAGUG…GCACCGAGUCGGUGCGCUAUUUUUU CXXCCCACAGGAAACCCAGGCUGGUUGGAGGGGUUU - 3’ (SEQ ID NO: 93)

[0125] Bold: Spacer Binding Sequence

[0126] Italic: Reverse Transcriptase Template

[0127] Underline: Primer binding site

[0128] Bold Italic: dinucleotide mutation to block exon 7 splicing; XX can be GG or AA. TALENs

[0129] The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right-TALEN, which references the handedness of DNA or a TALEN-pair.

[0130] The cipher for TALs has been reported (PCT Publication WO 2011/072246) wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence. The residues may be assembled to target a DNA sequence. In brief, a target site for binding of a TALEN is determined and a fusion molecule comprising a nuclease and a TAL effector protein. The TAL effector protein has a DNA binding domain that consists of consensus repeat sequences. Within the consensus repeat sequences at positions 12 and 13 within each repeat, the“repeat variable di-residue” (RVD), specifics the target, one RVD to one nucleotide (Cermak et al. Nucleic Acid Res, 2011 Jul: 39(12): e82). Upon binding, the nuclease cleaves the DNA so that cellular repair machinery can operate to make a genetic modification at the cut ends. The term TALEN means a protein comprising a Transcription Activator-like (TAL) effector binding domain and a nuclease domain and includes monomeric TALENs that are functional per se as well as others that require dimerization with another monomeric TALEN. The dimerization can result in a homodimeric TALEN when both monomeric TALEN are identical or can result in a heterodimeric TALEN when monomeric TALEN are different. TALENs have been shown to induce gene modification in immortalized human cells by means of the two-major eukaryotic DNA repair pathways, non-homologous end joining (NHEJ) and homology directed repair. TALENs are often used in pairs but monomeric TALENs are known. Cells for treatment by TALENs (and other genetic tools) include a cultured cell, an immortalized cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, a blastocyst, or a stem cell. In some embodiments, a TAL effector can be used to target other protein domains (e.g., non-nuclease protein domains) to specific nucleotide sequences. For example, a TAL effector can be linked to a protein domain from, without limitation, a DNA 20 interacting enzyme (e.g., a methylase, a topoisomerase, an integrase, a transposase, or a ligase), a transcription activators or repressor, or a protein that interacts with or modifies other proteins such as histones. Applications of such TAL effector fusions include, for example, creating or modifying epigenetic regulatory elements, making site- specific insertions, deletions, or repairs in DNA, controlling gene expression, and modifying chromatin structure.

[0131] The term nuclease includes exonucleases and endonucleases. The term endonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Non-limiting examples of endonucleases include type II restriction endonucleases such as FokI, HhaI, HindlII, NotI, BbvCl, EcoRI, BglII, and AlwI. Endonucleases also comprise rare-cutting endonucleases, typically having a polynucleotide recognition site of about 12-45 basepairs (bp) in length, more preferably of 14-45 bp. Rare-cutting endonucleases induce DNA double-strand breaks (DSBs) at a defined locus. Rare-cutting endonucleases can for example be a targeted endonuclease, a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as FokI or a chemical endonuclease. In chemical endonucleases, a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences. Such chemical endonucleases are comprised in the term“endonuclease” according to the present invention. Examples of such endonuclease include I-See I, I-Chu L I-Cre I, I-Csm I, PI-See L PI-Tti L PI-Mtu I, I-Ceu I, I-See IL 1- See III, HO, PI-Civ I, PI-Ctr L PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra L PI-Mav L PI-Meh I, PI-Mfu L PI-Mfl I, PI-Mga L PI-Mgo I, PI-Min L PI-Mka L PI-Mle I, PI-Mma I, PI- 30 Msh L PI- Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu L PI-Rma I, PI-Spb I, PI-Ssp L PI-Fae L PI-Mja I, PI-Pho L PI-Tag L PI-Thy I, PI-Tko I, PI-Tsp I, I-MsoI.

[0132] A genetic modification made by TALENs or other tools may be, for example, chosen from the list consisting of an insertion, a deletion, insertion of an exogenous nucleic acid fragment, and a substitution. The term insertion is used broadly to mean either literal insertion into the chromosome or use of the exogenous sequence as a template for repair. In general, a target DNA site is identified, and a TALEN-pair is created that will specifically bind to the site. The TALEN is delivered to the cell or embryo, e.g., as a protein, mRNA or by a vector that encodes the TALEN. The TALEN cleaves the DNA to make a double-strand break that is then repaired, often resulting in the creation of an indel, or incorporating sequences or polymorphisms contained in an accompanying exogenous nucleic acid that is either inserted into the chromosome or serves as a template for repair of the break with a modified sequence. This template-driven repair is a useful process for changing a chromosome and provides for effective changes to cellular chromosomes.

[0133] The term exogenous nucleic acid means a nucleic acid that is added to the cell or embryo, regardless of whether the nucleic acid is the same or distinct from nucleic acid sequences naturally in the cell. The term nucleic acid fragment is broad and includes a chromosome, expression cassette, gene, DNA, RNA, mRNA, or portion thereof. The cell or embryo may be, for instance, chosen from the group consisting non-human vertebrates, non-human primates, cattle, horse, swine, sheep, chicken, avian, rabbit, goats, dog, cat, laboratory animal, and fish.

[0134] Some embodiments involve a composition or a method of making a genetically modified livestock and/or artiodactyl comprising introducing a TALEN-pair into livestock and/or an artiodactyl cell or embryo that makes a genetic modification to DNA of the cell or embryo at a site that is specifically bound by the TALEN-pair and producing the livestock animal/artiodactyl from the cell. Direct injection may be used for the cell or embryo, e.g., into a zygote, blastocyst, or embryo. Alternatively, the TALEN and/or other factors may be introduced into a cell using any of many known techniques for introduction of proteins, RNA, mRNA, DNA, or vectors. Genetically modified animals may be made from the embryos or cells according to known processes, e.g., implantation of the embryo into a gestational host, or various cloning methods. The phrase“a genetic modification to DNA of the cell at a site that is specifically bound by the TALEN”, or the like, means that the genetic modification is made at the site cut by the nuclease on the TALEN when the TALEN is specifically bound to its target site. The nuclease does not cut exactly where the TALEN-pair binds, but rather at a defined site between the two binding sites.

[0135] Some embodiments involve a composition or a treatment of a cell that is used for cloning the animal. The cell may be a livestock and/or artiodactyl cell, a cultured cell, a primary cell, a primary somatic cell, a zygote, a germ cell, a primordial germ cell, or a stem cell. For example, an embodiment is a composition or a method of creating a genetic modification comprising exposing a plurality of primary cells in a culture to TALEN proteins or a nucleic acid encoding a TALEN or TALENs. The TALENs may be introduced as proteins or as nucleic acid fragments, e.g., encoded by mRNA or a DNA sequence in a vector. Zinc Finger Nucleases

[0136] Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences, and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to alter the genomes of higher organisms. ZFNs may be used in method of inactivating genes.

[0137] A zinc finger DNA-binding domain has about 30 amino acids and folds into a stable structure. Each finger primarily binds to a triplet within the DNA substrate. Amino acid residues at key positions contribute to most of the sequence-specific interactions with the DNA site. These amino acids can be changed while maintaining the remaining amino acids to preserve the necessary structure. Binding to longer DNA sequences is achieved by linking several domains in tandem. Other functionalities like non-specific FokI cleavage domain (N), transcription activator domains (A), transcription repressor domains I and methylases (M) can be fused to a ZFPs to form ZFNs respectively, zinc finger transcription activators (ZFA), zinc finger transcription repressors (ZFR, and zinc finger methylases (ZFM). Vectors and Nucleic acids

[0138] A variety of nucleic acids may be introduced into cells, for knockout purposes, for inactivation of a gene, to obtain expression of a gene, or for other purposes. As used herein, the term nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double- stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.

[0139] The target nucleic acid sequence can be operably linked to a regulatory region such as a promoter. Regulatory regions can be porcine regulatory regions or can be from other species. As used herein, operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid.

[0140] In general, any type of promoter can be operably linked to a target nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, inducible promoters, and promoters responsive or unresponsive to a particular stimulus. In some embodiments, a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used (i.e., a constitutive promoter). For example, a beta-actin promoter such as the chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or 3- phosphoglycerate kinase (PGK) promoter can be used, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter. In some embodiments, a fusion of the chicken beta actin gene promoter and the CMV enhancer is used as a promoter.

[0141] Additional regulatory regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, can sometimes be obtained without such additional elements.

[0142] A nucleic acid construct may be used that encodes signal peptides or selectable expressed markers. Signal peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.

[0143] In some embodiments, a sequence encoding a selectable marker can be flanked by recognition sequences for a recombinase such as, e.g., Cre or Flp. For example, the selectable marker can be flanked by loxP recognition sites (34-bp recognition sites recognized by the Cre recombinase) or FRT recognition sites such that the selectable marker can be excised from the construct. A transposon containing a Cre- or Flp-activatable transgene interrupted by a selectable marker gene also can be used to obtain transgenic animals with conditional expression of a transgene. For example, a promoter driving expression of the marker/transgene can be either ubiquitous or tissue-specific, which would result in the ubiquitous or tissue-specific expression of the marker in F0 animals (e.g., pigs). Tissue specific activation of the transgene can be accomplished, for example, by crossing a pig that ubiquitously expresses a marker-interrupted transgene to a pig expressing Cre or Flp in a tissue-specific manner, or by crossing a pig that expresses a marker-interrupted transgene in a tissue-specific manner to a pig that ubiquitously expresses Cre or Flp recombinase. Controlled expression of the transgene or controlled excision of the marker allows expression of the transgene.

[0144] In some embodiments, the exogenous nucleic acid encodes a polypeptide. A nucleic acid sequence encoding a polypeptide can include a tag sequence that encodes a“tag” designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAG™ tag (Kodak, New Haven, CT).

[0145] Nucleic acid constructs can be introduced into embryonic, fetal, or adult artiodactyl/livestock cells of any type, including, for example, germ cells such as an oocyte or an egg, a progenitor cell, an adult or embryonic stem cell, a primordial germ cell, a kidney cell such as a PK-15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast, using a variety of techniques. Non-limiting examples of techniques include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells.

[0146] In transposon systems, the transcriptional unit of a nucleic acid construct, i.e., the regulatory region operably linked to an exogenous nucleic acid sequence, is flanked by an inverted repeat of a transposon. Several transposon systems, including, for example, Sleeping Beauty , Frog Prince , Tol2 , Minos , Hsmar1 , and Passport have been developed to introduce nucleic acids into cells, including mice, human, and pig cells. The Sleeping Beauty transposon is particularly useful. A transposase can be delivered as a protein, encoded on the same nucleic acid construct as the exogenous nucleic acid, can be introduced on a separate nucleic acid construct, or provided as an mRNA (e.g., an in vitro-transcribed and capped mRNA).

[0147] Nucleic acids can be incorporated into vectors. A vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA. A vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non-viral vectors (e.g., transposons) used for gene delivery in animals have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence. Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.

[0148] Many different types of vectors are known. For example, plasmids and viral vectors, e.g., retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5’ flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

[0149] As used herein, the term nucleic acid refers to both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, as well as naturally occurring and chemically modified nucleic acids, e.g., synthetic bases or alternative backbones. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand). The term transgenic is used broadly herein and refers to a genetically modified organism or genetically engineered organism whose genetic material has been altered using genetic engineering techniques. A knockout artiodactyl is thus transgenic regardless of whether or not exogenous genes or nucleic acids are expressed in the animal or its progeny. Genetically modified animals

[0150] Animals may be modified using TALENs or other genetic engineering tools, including recombinase fusion proteins, or various vectors that are known. A genetic modification made by such tools may comprise disruption of a gene. The term disruption of a gene refers to preventing the formation of a functional gene product. A gene product is functional only if it fulfills its normal (wild-type) functions. Disruption of the gene prevents expression of a functional factor encoded by the gene and comprises an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal. The disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene. Materials and methods of genetically modifying animals are further detailed in U.S.8,518,701; U.S.2010/0251395; and U.S.2012/0222143 which are hereby incorporated herein by reference for all purposes; in case of conflict, the instant specification is controlling. The term trans-acting refers to processes acting on a target gene from a different molecule (i.e., intermolecular). A trans-acting element is usually a DNA sequence that contains a gene. This gene codes for a protein (or microRNA or other diffusible molecule) that is used in the regulation of the target gene. The trans-acting gene may be on the same chromosome as the target gene, but the activity is via the intermediary protein or RNA that it encodes. Embodiments of trans-acting gene are, e.g., genes that encode targeting endonucleases. Inactivation of a gene using a dominant negative generally involves a trans-acting element. The term cis-regulatory or cis-acting means an action without coding for protein or RNA; in the context of gene inactivation, this generally means inactivation of the coding portion of a gene, or a promoter and/or operator that is necessary for expression of the functional gene.

[0151] Various techniques known in the art can be used to inactivate genes to make knock-out animals and/or to introduce nucleic acid constructs into animals to produce founder animals and to make animal lines, in which the knockout or nucleic acid construct is integrated into the genome. Such techniques include, without limitation, pronuclear microinjection, retrovirus mediated gene transfer into germ lines, gene targeting into embryonic stem cells, electroporation of embryos, sperm-mediated gene transfer, and in vitro transformation of somatic cells, such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transplantation. Pronuclear microinjection, sperm mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. An animal that is genomically modified is an animal wherein all of its cells have the genetic modification, including its germ line cells. When methods are used that produce an animal that is mosaic in its genetic modification, the animals may be inbred and progeny that are genomically modified may be selected. Cloning, for instance, may be used to make a mosaic animal if its cells are modified at the blastocyst state, or genomic modification can take place when a single-cell is modified. Animals that are modified so they do not sexually mature can be homozygous or heterozygous for the modification, depending on the specific approach that is used. If a particular gene is inactivated by a knock out modification, homozygosity would normally be required. If a particular gene is inactivated by an RNA interference or dominant negative strategy, then heterozygosity is often adequate.

[0152] Typically, in pronuclear microinjection, a nucleic acid construct is introduced into a fertilized egg; 1 or 2 cell fertilized eggs are used as the pronuclei containing the genetic material from the sperm head and the eggs are visible within the protoplasm. Pronuclear staged fertilized eggs can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of donor animals). In vitro fertilized eggs can be produced as follows. For example, swine ovaries can be collected at an abattoir, and maintained at 22-28°C during transport. Ovaries can be washed and isolated for follicular aspiration, and follicles ranging from 4-8 mm can be aspirated into 50 mL conical centrifuge tubes using 18-gauge needles and under vacuum. Follicular fluid and aspirated oocytes can be rinsed through pre-filters with commercial TL-HEPES (Minitube, Verona, WI). Oocytes surrounded by a compact cumulus mass can be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM (Minitube, Verona, WI) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid, 50 mM 2- mercaptoethanol, 0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for approximately 22 hours in humidified air at 38.7°C and 5% CO ₂ . Subsequently, the oocytes can be moved to fresh TCM-199 maturation medium, which will not contain cAMP, PMSG or hCG and incubated for an additional 22 hours. Matured oocytes can be stripped of their cumulus cells by vortexing in 0.1% hyaluronidase for 1 minute.

[0153] For swine, mature oocytes can be fertilized in 500 ml Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, WI) in Minitube 5-well fertilization dishes. In preparation for in vitro fertilization (IVF), freshly-collected or frozen boar semen can be washed and resuspended in PORCPRO IVF Medium to 4 x 10 ⁵ sperm. Sperm concentrations can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, WI). Final in vitro insemination can be performed in a 10ml volume at a final concentration of approximately 40 motile sperm/oocyte, depending on boar. Incubate all fertilizing oocytes at 38.7°C in 5.0% CO2 atmosphere for 6 hours. Six hours post-insemination, presumptive zygotes can be washed twice in NCSU-23 and moved to 0.5 mL of the same medium. This system can produce 20-30% blastocysts routinely across most boars with a 10-30% polyspermic insemination rate.

[0154] Linearized nucleic acid constructs can be injected into one of the pronuclei. Then the injected eggs can be transferred to a recipient female (e.g., into the oviducts of a recipient female) and allowed to develop in the recipient female to produce the transgenic animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 X g for 5 minutes to sediment lipids allowing visualization of the pronucleus. The embryos can be injected using an Eppendorf FEMTOJET injector and can be cultured until blastocyst formation. Rates of embryo cleavage and blastocyst formation and quality can be recorded.

[0155] Embryos can be surgically transferred into uteri of asynchronous recipients. Typically, 100-200 (e.g., 150-200) embryos can be deposited into the ampulla-isthmus junction of the oviduct using a 5.5-inch TOMCAT ^® catheter. After surgery, real-time ultrasound examination of pregnancy can be performed.

[0156] In somatic cell nuclear transfer, a transgenic artiodactyl cell (e.g., a transgenic pig cell or bovine cell) such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell that includes a nucleic acid construct described above, can be introduced into an enucleated oocyte to establish a combined cell. Oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area. Typically, an injection pipette with a sharp beveled tip is used to inject the transgenic cell into an enucleated oocyte arrested at meiosis 2. In some conventions, oocytes arrested at meiosis-2 are termed eggs. After producing a porcine or bovine embryo (e.g., by fusing and activating the oocyte), the embryo is transferred to the oviducts of a recipient female, about 20 to 24 hours after activation. For pigs, recipient females can be checked for pregnancy approximately 20-21 days after transfer of the embryos.

[0157] Standard breeding techniques can be used to create animals that are homozygous for the exogenous nucleic acid from the initial heterozygous founder animals. Homozygosity may not be required, however. Transgenic pigs described herein can be bred with other pigs of interest.

[0158] In some embodiments, a nucleic acid of interest and a selectable marker can be provided on separate transposons and provided to either embryos or cells in unequal amount, where the amount of transposon containing the selectable marker far exceeds (5-10-fold excess) the transposon containing the nucleic acid of interest. Transgenic cells or animals expressing the nucleic acid of interest can be isolated based on presence and expression of the selectable marker. Because the transposons will integrate into the genome in a precise and unlinked way (independent transposition events), the nucleic acid of interest and the selectable marker are not genetically linked and can easily be separated by genetic segregation through standard breeding. Thus, transgenic animals can be produced that are not constrained to retain selectable markers in subsequent generations, an issue of some concern from a public safety perspective.

[0159] Once transgenic animals have been generated, expression of an exogenous nucleic acid can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether or not integration of the construct has taken place. Polymerase chain reaction (PCR) techniques also can be used in the initial screening. PCR refers to a procedure or technique in which target nucleic acids are amplified. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length but can range from 10 nucleotides to hundreds of nucleotides in length. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplified. At the blastocyst stage, embryos can be individually processed for analysis by PCR, Southern hybridization and splinkerette PCR

[0160] Expression of a nucleic acid sequence encoding a polypeptide in the tissues of transgenic pigs can be assessed using techniques that include, for example, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, Western analysis, immunoassays such as enzyme-linked immunosorbent assays, and reverse-transcriptase PCR (RT-PCR). Interfering RNAs

[0161] A variety of interfering RNA (RNAi) are known. Double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous gene transcripts. RNA-induced silencing complex (RISC) metabolizes dsRNA to small 21-23-nucleotide small interfering RNAs (siRNAs). RISC contains a double stranded RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut 2 or Ago2). RISC utilizes antisense strand as a guide to find a cleavable target. Both siRNAs and microRNAs (miRNAs) are known. A method of disrupting a gene in a genetically modified animal comprises inducing RNA interference against a target gene and/or nucleic acid such that expression of the target gene and/or nucleic acid is reduced.

[0162] For example, the exogenous nucleic acid sequence can induce RNA interference against a nucleic acid encoding a polypeptide. For example, double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) homologous to a target DNA can be used to reduce expression of that DNA. In general, shRNAs are transcribed as a single-stranded RNA molecule containing complementary regions, which can anneal and form short hairpins.

[0163] The probability of finding a single, individual functional siRNA or miRNA directed to a specific gene is high. The predictability of a specific sequence of siRNA, for instance, is about 50% but a number of interfering RNAs may be made with good confidence that at least one of them will be effective.

[0164] Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that express an RNAi directed against a gene, e.g., a gene selective for a developmental stage. The RNAi may be, for instance, selected from the group consisting of siRNA, shRNA, dsRNA, RISC and miRNA. Inducible systems

[0165] An inducible system may be used to control expression of a gene. Various inducible systems are known that allow spatiotemporal control of expression of a gene. Several have been proven to be functional in vivo in transgenic animals. The term inducible system includes traditional promoters and inducible gene expression elements.

[0166] An example of an inducible system is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of the nucleic acid. In this system, a mutated Tet repressor (TetR) is fused to the activation domain of herpes simplex virus VP16 trans-activator protein to create a tetracycline-controlled transcriptional activator (tTA), which is regulated by tet or doxycycline (dox). In the absence of antibiotic, transcription is minimal, while in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone or rapamycin systems. Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor and the product of the ultraspiracle gene (USP). Expression is induced by treatment with ecdysone or an analog of ecdysone such as muristerone A. The agent that is administered to the animal to trigger the inducible system is referred to as an induction agent.

[0167] The tetracycline-inducible system and the Cre/loxP recombinase system (either constitutive or inducible) are among the more commonly used inducible systems. The tetracycline-inducible system involves a tetracycline-controlled transactivator (tTA)/ reverse tTA (rtTA). A method to use these systems in vivo involves generating two lines of genetically modified animals. One animal line expresses the activator (tTA, rtTA, or Cre recombinase) under the control of a selected promoter. Another set of transgenic animals express the acceptor, in which the expression of the gene of interest (or the gene to be modified) is under the control of the target sequence for the tTA/rtTA transactivators (or is flanked by loxP sequences). Mating the two strains of mice provides control of gene expression.

[0168] The tetracycline-dependent regulatory systems (tet systems) rely on two components, i.e., a tetracycline-controlled transactivator (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner. In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is termed tet-OFF, because tetracycline or doxycycline allows transcriptional down- regulation. Administration of tetracycline or its derivatives allows temporal control of transgene expression in vivo. rtTA is a variant of tTA that is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation. This tet system is therefore termed tet- ON. The tet systems have been used in vivo for the inducible expression of several transgenes, encoding, e.g., reporter genes, oncogenes, or proteins involved in a signaling cascade.

[0169] The Cre/lox system uses the Cre recombinase, which catalyzes site-specific recombination by crossover between two distant Cre recognition sequences, i.e., loxP sites. A DNA sequence introduced between the two loxP sequences (termed floxed DNA) is excised by Cre-mediated recombination. Control of Cre expression in a transgenic animal, using either spatial control (with a tissue- or cell-specific promoter) or temporal control (with an inducible system), results in control of DNA excision between the two loxP sites. One application is for conditional gene inactivation (conditional knockout). Another approach is for protein over-expression, wherein a floxed stop codon is inserted between the promoter sequence and the DNA of interest. Genetically modified animals do not express the transgene until Cre is expressed, leading to excision of the floxed stop codon. This system has been applied to tissue-specific oncogenesis and controlled antigen receptor expression in B lymphocytes. Inducible Cre recombinases have also been developed. The inducible Cre recombinase is activated only by administration of an exogenous ligand. The inducible Cre recombinases are fusion proteins containing the original Cre recombinase and a specific ligand-binding domain. The functional activity of the Cre recombinase is dependent on an external ligand that is able to bind to this specific domain in the fusion protein.

[0170] Embodiments include an in vitro cell, an in vivo cell, and a genetically modified animal such as a livestock animal that comprise a gene under control of an inducible system. The genetic modification of an animal may be genomic or mosaic. The inducible system may be, for instance, selected from the group consisting of Tet-On, Tet-Off, Cre-lox, and Hif1alpha. An embodiment is a gene set forth herein. Dominant Negatives

[0171] Genes may thus be disrupted not only by removal or RNAi suppression but also by creation/expression of a dominant negative variant of a protein which has inhibitory effects on the normal function of that gene product. The expression of a dominant negative (DN) gene can result in an altered phenotype, exerted by a) a titration effect; the DN PASSIVELY competes with an endogenous gene product for either a cooperative factor or the normal target of the endogenous gene without elaborating the same activity, b) a poison pill (or monkey wrench) effect wherein the dominant negative gene product ACTIVELY interferes with a process required for normal gene function, c) a feedback effect, wherein the DN ACTIVELY stimulates a negative regulator of the gene function. Founder animals, animal lines, traits, and reproduction

[0172] Founder animals (F0 generation) may be produced by cloning and other methods described herein. The founders can be homozygous for a genetic modification, as in the case where a zygote or a primary cell undergoes a homozygous modification. Similarly, founders can also be made that are heterozygous. The founders may be genomically modified, meaning that the cells in their genome have undergone modification. Founders can be mosaic for a modification, as may happen when vectors are introduced into one of a plurality of cells in an embryo, typically at a blastocyst stage. Progeny of mosaic animals may be tested to identify progeny that are genomically modified. An animal line is established when a pool of animals has been created that can be reproduced sexually or by assisted reproductive techniques, with heterogeneous or homozygous progeny consistently expressing the modification.

[0173] In livestock, many alleles are known to be linked to various traits such as production traits, type traits, workability traits, and other functional traits. Artisans are accustomed to monitoring and quantifying these traits An animal line may include a trait chosen from a trait in the group consisting of a production trait, a type trait, a workability trait, a fertility trait, a mothering trait, and a disease resistance trait. Further traits include expression of a recombinant gene product. Recombinases

[0174] Embodiments of the invention include administration of a targeted nuclease system with a recombinase (e.g., a RecA protein, a Rad51) or other DNA-binding protein associated with DNA recombination. A recombinase forms a filament with a nucleic acid fragment and, in effect, searches cellular DNA to find a DNA sequence substantially homologous to the sequence. For instance, a recombinase may be combined with a nucleic acid sequence that serves as a template for HDR. The recombinase is then combined with the HDR template to form a filament and placed into the cell. The recombinase and/or HDR template that combines with the recombinase may be placed in the cell or embryo as a protein, an mRNA, or with a vector that encodes the recombinase. The disclosure of U.S. 2011/0059160 (U.S. Patent Application No. 12/869,232) is hereby incorporated herein by reference for all purposes; in case of conflict, the specification is controlling. The term recombinase refers to a genetic recombination enzyme that enzymatically catalyzes, in a cell, the joining of relatively short pieces of DNA between two relatively longer DNA strands. Recombinases include Cre recombinase, Hin recombinase, RecA, RAD51, Cre, and FLP. Cre recombinase is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria Salmonella. Hin belongs to the serine recombinase family of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination. RAD51 is a human gene. The protein encoded by this gene is a member of the RAD51 protein family which assists in repair of DNA double strand breaks. RAD51 family members are homologous to the bacterial RecA and yeast Rad51. Cre recombinase is an enzyme that is used in experiments to delete specific sequences that are flanked by loxP sites. FLP refers to Flippase recombination enzyme (FLP or Flp) derived from the 2µ plasmid of the baker’s yeast Saccharomyces cerevisiae.

[0175] Herein,“RecA” or“RecA protein” refers to a family of RecA-like recombination proteins having essentially all or most of the same functions, particularly: (i) the ability to position properly oligonucleotides or polynucleotides on their homologous targets for subsequent extension by DNA polymerases; (ii) the ability topologically to prepare duplex nucleic acid for DNA synthesis; and, (iii) the ability of RecA/oligonucleotide or RecA/polynucleotide complexes efficiently to find and bind to complementary sequences. The best characterized RecA protein is from E. coli; in addition to the original allelic form of the protein, a number of mutant RecA-like proteins have been identified, for example, RecA803. Further, many organisms have RecA-like strand-transfer proteins including, for example, yeast, Drosophila, mammals including humans, and plants. These proteins include, for example, Rec1, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2 and DMC1. An embodiment of the recombination protein is the RecA protein of E. coli. Alternatively, the RecA protein can be the mutant RecA-803 protein of E. coli, a RecA protein from another bacterial source or a homologous recombination protein from another organism. Compositions and kits

[0176] The present invention also provides compositions and kits containing, for example, nucleic acid molecules encoding site-specific endonucleases, base editor (e.g. ABE, CBE), CRISPR, Cas9, ZNFs, TALENs, RecA-gal4 fusions, polypeptides of the same, compositions containing such nucleic acid molecules or polypeptides, or engineered cell lines. An HDR may also be provided that is effective for introgression of an indicated allele. Such items can be used, for example, as research tools, or therapeutically. Short Homology Arm HDR (SHA-HDR)

[0177] In double stranded SHA-HDR a template, contained within a plasmid, is introduced into the cell at about the same time as a nuclease. The template is liberated from the plasmid by the introduction of an appropriate restriction enzyme at about the same time. In some embodiments, the insert is liberated from the plasmid by cas9 endonuclease. While the exact mechanism by which HDR is introduced into a genome by the cell is unknown, the inventors’ experiments show that double stranded DNA when provided at about the same time as a targeted double stranded break (DSB) is made requires less template and requires much shorter homology arms than an ssODN template. In these examples, those of skill in the art will appreciate that that is a range of ratios of nuclease to plasmid to enzyme that can be empirically validated to achieve optimum HDR and that, in some instances, the ratio is determined by the size of the template that is used to make the deletion or insertion edited into the genome.

[0178] FIG.1 is a cartoon illustrating the CD163 gene. Illustrated are the 16 exons with exon 16 being intracellular. Also, shown are the scavenger receptor cysteine-rich (SRCR) domains. As shown, some of the SRCR domains are shared between multiple exons with SCRC 1 being shared by exons 2, 3 and 4 and SRCR6 being shared between exons 7, 8 and 9, SRCR7 shared between exons 9 and, 10 and 11 and SRCR9 being shared between exons 12 and 13. CD163 has been described as having a variety of important biological functions, including as an erythroblast binding factor, enhancing the survival, proliferation and differentiation of immature erythroblasts, through association with SRCR domain 2 and CD163-expressing macrophages also clear senescent and malformed erythroblasts. SRCR domain 3 plays a crucial role as a hemoglobin (Hb)- haptoglobin (Hp) scavenger receptor. Free Hb is oxidative and toxic; once complexed with Hp it is cleared through binding to SRCR3 on the surface of macrophages and subsequent endocytosis. This prevents oxidative damage, maintains homeostasis, and aids the recycling of iron. Recently, CD163 was also shown to interact with HMGB1-haptoglobin complexes and regulate the inflammatory response in a heme-oxygenase 1 (HO-1) dependent manner. CD163-expressing macrophages were also found to be involved in the clearance of a cytokine named TNF-like weak inducer of apoptosis (TWEAK), with all SRCRs apart from SRCR5 being involved in this process. Soluble CD163 can be found at a high concentration in blood plasma but its function in this niche is still partially unknown. The interaction of CD163 can also be involved in ischaemic injury tissue regeneration. Thus, it seems, that to maintain the greatest physiological functions it is important to modify the CD163 protein as little as possible.

[0179] CD163 is a 3,400 bp mRNA transcript coding for a protein of 1,110 aa. FIG.3 and FIG. 4. An alignment of swine, human, mouse and monkey CD163 shows a highly conserved protein with only several mismatches throughout its sequence. The SRCR5 from CD163 has been crystalized. A long loop region with a special electrostatic potential was identified, and it has been hypothesized that Arg561 in the long loop region is important for PRRSV binding and infection. However, it should be noted that a separate macrophage surface receptor CD169 has also been found to bind PRRSV and internalize the virus. It appears, however, that virus internalized by CD169 is not uncoated and is not infective. Overexpression of CD169 in non-permissive PK-15 cells showed internalization but not productive replication of PRRSV while overexpression of cd163 rendered non-susceptible cells permissive to PRRSV. (Burkard et al. pg. 3 2017). These results indicate, that binding of the virus to the receptor is not dispositive of infectivity. Consequently, there may be multiple domains of SRCR5 which modulate one or more of binding, internalization and uncoating of the virus. Therefore, the inventors hypothesized that a truncation of the CD163 gene to delete, in-frame, amino acid residues of the protein that are unique to PRRSV susceptible animals will provide PRRSV resistant animals that maintain the greatest functionality of the conserved Sus scrofa CD163 SRCR5 motif.

[0180] Various exemplary embodiments of devices and compounds as generally described above and methods according to this invention, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention in any fashion.

EXAMPLES

[0181] TABLE I provides a listing of TALENs and their sequences used in the following examples.

[0182] TABLE I

[0183] When used with a CRISPR Cas9 nuclease or a base editor (e.g., ABE, CBE) the following gRNAs are used

EXAMPLE 1

[0184] DELETION OF E537-D558 AND A UNIQUE SWINE AMINO ACID USING AN ssODN TEMPLATE

[0185] In one embodiment for limiting or eliminating the infectivity of PRRSV, the residues E537- D558 are deleted (FIG.4). This is a 66 bp deletion and a 22 aa deletion. An ssODN HDR template was designed that could be used to make this deletion may comprise the following sequence:

[0186] 5’

CTGGGGGGAGCTCACTTTGGAGAAGGAAGTGGACAGATCTGGGCTGGGACAT GTA GCCACAGCAGGGACGTCGGCGTAGTCTGCTCAAGT 3’. (SEQ ID NO: 22) [0187] However, it is noted that the HDR sequence may be longer or shorter in one or both of the arms than that provided herein. TALENs (FIG.4), TALENS ssCD1637.5 L/R, ssCD1637.6 L/R, ssCD163 7.7 L/R were designed to make an appropriate double stranded break. When using a CRISPr/CAS9 nuclease system gRNAs used include: ssCD163 g7.4, ssCD163, g7.5.

[0188] The final sequence of the edited protein is shown below where the sequence EEFQCEGHESHLSLCPVAPRPD (SEQ ID NO: 23) is deleted in-frame between the Alanine and Glycine residues shaded in gray below.

[0189] MVLLEDSGSADFRRCSAHLSSFTFAVVAVLSACLVTSSLGGKDKELRLTGGENK CSGRVEVKVQEEWGTVCNNGWDMDVVSVVCRQLGCPTAIKATGWANFSAGSGRIWM DHVSCRGNESALWDCKHDGWGKHNCTHQQDAGVTCSDGSDLEMRLVNGGNRCLGRI EVKFQERWGTVCDDNFNINHASVVCKQLECGSAVSFSGSANFGEGSGPIWFDDLVCNG NESALWNCKHEGWGKHNCDHAEDAGVICLNGADLKLRVVDGLTECSGRLEVKFQGE WGTICDDGWDSDDAAVACKQLGCPTAVTAIGRVNASEGTGHIWLDSVSCHGHESALW QCRHHEWGKHYCNHNEDAGVTCSDGSDLELRLKGGGSHCAGTVEVEIQKLVGKVCDR SWGLKEADVVCRQLGCGSALKTSYQVYSKTKATNTWLFVSSCNGNETSLWDCKNWQ WGGLSCDHYDEAKITCSAHRKPRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASV LCRELQCGTVVSLLGGAHFGEGSGQIWAGTCSHSRDVGVVCSRYTQIRLVNGKTPCEG RVELNILGSWGSLCNSHWDMEDAHVLCQQLKCGVALSIPGGAPFGKGSEQVWRHMFH CTGTEKHMGDCSVTALGASLCSSGQVASVICSGNQSQTLSPCNSSSSDPSSSIISEESGV A CIGSGQLRLVDGGGRCAGRVEVYPGASWGTICDDSWDLNDAHVVCKQLSCGWAINAT GSAHFGEGTGPIWLDEINCNGKESHIWQCHSHGWGRHNCRHKEDAGVICSEFMSLRLIS ENSRETCAGRLEVFYNGAWGSVGRNSMSPATVGVVCRQLGCADRGDISPASSDKTVSR HMWVDNVQCPKGPDTLWQCPSSPWKKRLASPSEETWITCANKIRLQEGNTNCSGRVEI WYGGSWGTVCDDSWDLEDAQVVCRQLGCGSALEAGKEPAFGQGTGPIWLNEVKCKG NEPSLWDCPARSWGHSDCGHKEDAAVTCSEIAKSRESLHATGRSSFVALAIFGVILLACL IAFLIWTQKRRQRQRLSVFSGGENSVHQIQYREMNSCLKADETDMLNPSGDHSEVQ

(SEQ ID NO: 24)

EXAMPLE 2

[0190] DELETION OF E537-D558 ANE A UNIQUE SWINE AMINO ACID USING A SHORT HOMOLOGY ARM ds TEMPLATE

[0191] In this example, the same deletion is made as in Example 1, however, instead of a ssODN template, a double stranded DNA template is made and is introduced into the cell along with an appropriate restriction nuclease to liberate the template from the plasmid. In this example, the construct is shown in FIG.5. In this example, the template is liberated from the plasmid by the CAS 9 endonuclease. However, those of skill in the art will appreciate that the template can be liberated from the plasmid with any nuclease or restriction enzyme useful.

[0192] In this example the following construct can be used:

[0193] GGGAGGCGTTCGGGCCACAGcggTGATCTCCTGGGGGGAGCTCACTTTGGAGAA GGAAGTGGACAGATCTGGGCTGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGTCT GCTCAAGTGAGGCTccaCTGTGGCCCGAACGCCTCCC (SEQ ID NO: 25)

[0194] Underlined: Universal gRNA

[0195] Italic: 5’ HR ARM

[0196] Bold Italic: 3’ HR Arm

[0197] Lower case: PAM sequence

[0198] As used in Example 2, the edited protein has the same sequence as that in EXAMPLE 1 (SEQ ID NO: 24)

EXAMPLE 3

[0199] REMOVAL of E537-V567 and a UNIQUE SWNE AMINO ACID USING AN ssODN TEMPLATE

[0200] In another embodiment for limiting or eliminating the infectivity of PRRSV, the residues E537- V567 are deleted (FIG.6). This is a 31aa deletion and a 93bp deletion from the gene. In this example, one HDR template useful to make this deletion may comprise the following sequence:

[0201] 5’CTGGGGGGAGCTCACTTTGGAGAAGGAAGTGGACAGATCTGGGCT

GGCGTAGTCTGCTCAAGTGAGACCCAGGGAATGTGTTCACTTTGT 3’. (SEQ ID NO: 26)

[0202] However, it is noted that the HDR sequence may be longer or shorter in one or both of the arms than that provided herein. TALENs useful in this example include: ssCD163 7.5 L/R, ssCD1637.6 L/R, ssCD1637.7 L/R. When CRISPR/CAS9 is used, guide RNA includes: gRNAs: ssCD163 g7.4, ssCD163, g7.5. The edited protein has the following sequence where EEFQCEGHESHLSLCPVAPRPDGTCSHSRDV (SEQ ID NO: 28) is deleted between the alanine (A) and glycine (G) residues shaded in gray:

[0203] MVLLEDSGSADFRRCSAHLSSFTFAVVAVLSACLVTSSLGGKDKELRLTGGENK CSGRVEVKVQEEWGTVCNNGWDMDVVSVVCRQLGCPTAIKATGWANFSAGSGRIWM DHVSCRGNESALWDCKHDGWGKHNCTHQQDAGVTCSDGSDLEMRLVNGGNRCLGRI EVKFQERWGTVCDDNFNINHASVVCKQLECGSAVSFSGSANFGEGSGPIWFDDLVCNG NESALWNCKHEGWGKHNCDHAEDAGVICLNGADLKLRVVDGLTECSGRLEVKFQGE WGTICDDGWDSDDAAVACKQLGCPTAVTAIGRVNASEGTGHIWLDSVSCHGHESALW QCRHHEWGKHYCNHNEDAGVTCSDGSDLELRLKGGGSHCAGTVEVEIQKLVGKVCDR SWGLKEADVVCRQLGCGSALKTSYQVYSKTKATNTWLFVSSCNGNETSLWDCKNWQ WGGLSCDHYDEAKITCSAHRKPRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASV LCRELQCGTVVSLLGGAHFGEGSGQIWAGVVCSRYTQIRLVNGKTPCEGRVELNILGSW GSLCNSHWDMEDAHVLCQQLKCGVALSIPGGAPFGKGSEQVWRHMFHCTGTEKHMG DCSVTALGASLCSSGQVASVICSGNQSQTLSPCNSSSSDPSSSIISEESGVACIGSGQLR LV DGGGRCAGRVEVYPGASWGTICDDSWDLNDAHVVCKQLSCGWAINATGSAHFGEGTG PIWLDEINCNGKESHIWQCHSHGWGRHNCRHKEDAGVICSEFMSLRLISENSRETCAGR LEVFYNGAWGSVGRNSMSPATVGVVCRQLGCADRGDISPASSDKTVSRHMWVDNVQC PKGPDTLWQCPSSPWKKRLASPSEETWITCANKIRLQEGNTNCSGRVEIWYGGSWGTV CDDSWDLEDAQVVCRQLGCGSALEAGKEPAFGQGTGPIWLNEVKCKGNEPSLWDCPA RSWGHSDCGHKEDAAVTCSEIAKSRESLHATGRSSFVALAIFGVILLACLIAFLIWTQKR RQRQRLSVFSGGENSVHQIQYREMNSCLKADETDMLNPSGDHSEVQ (SEQ ID NO: 27) EXAMPLE 4

[0204] REMOVAL of E537-V567 and a UNIQUE SWINE AMINO ACID USING A dsDNA TEMPLATE

[0205] In this example, the same deletion is made as in Example 3, however, instead of a ssODN template, a double stranded DNA template is made and is introduced into the cell along with an appropriate restriction nuclease to liberate the template from the plasmid. In this example, the construct is shown in FIG.7. In this example, the template is liberated from the plasmid by the CAS9 endonuclease.

[0206] In this example the following construct is used:

[0207] GGGAGGCGTTCGGGCCACAGcggTGATCTCCTGGGGGGAGCTCACTTTGGAGAA GGAAGTGGACAGATCTGGGCTGGCGTAGTCTGCTCAAGTGAGACCCAGGGAATGTGTT CACTTTGTTCCGCTccaCTGTGGCCCGAACGCCTCCC (SEQ ID NO: 29)

[0208] Underlined: Universal gRNA

[0209] Italic: 5’ HR ARM

[0210] Bold Italic: 3’ HR Arm

[0211] Lower case: PAM sequence

[0212] In this Example, the final sequence is the same as that shown in EXAMPLE 3 (SEQ ID NO: 27). EXAMPLE 5

[0213] REMOVAL OF S520-C571 and THREE UNIQUE SWINE AMINO ACIDS USING AN ssODN TEMPLATE

[0214] In this example, a 52 aa deletion is made comprising a 156bp deletion from the gene (FIG. 8). one HDR template useful to make this deletion may comprise the following sequence:

[0215] 5’

GCGGCCAGCGTGCTGTGCAGGGAACTACAGTGCGGCACTGTGGTTTCAAGTGAGA CCAGGGAAT GTGTTCACTTTGTTCCCATGCCATG 3’ (SEQ ID NO: 30)

[0216] However, it is noted that the HDR sequence may be longer or shorter in one or both of the arms than that provided herein. TALENs useful to make this edit include ssCD163 7.5 L/R, ssCD163 7.6 L/R, ssCD163 7.7 L/R. When CRISPR/CAS9 is used, the following guide RNAs may be used: ssCD163 g7.4, ssCD163, g7.5

[0217] The edited protein has the following sequence after deletion of S520-C571: SLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVC (SEQ ID NO: 31) between the valine (V) and serine (S) residues highlighted in gray below.

[0218] MVLLEDSGSADFRRCSAHLSSFTFAVVAVLSACLVTSSLGGKDKELRLTGGENK CSGRVEVKVQEEWGTVCNNGWDMDVVSVVCRQLGCPTAIKATGWANFSAGSGRIWM DHVSCRGNESALWDCKHDGWGKHNCTHQQDAGVTCSDGSDLEMRLVNGGNRCLGRI EVKFQERWGTVCDDNFNINHASVVCKQLECGSAVSFSGSANFGEGSGPIWFDDLVCNG NESALWNCKHEGWGKHNCDHAEDAGVICLNGADLKLRVVDGLTECSGRLEVKFQGE WGTICDDGWDSDDAAVACKQLGCPTAVTAIGRVNASEGTGHIWLDSVSCHGHESALW QCRHHEWGKHYCNHNEDAGVTCSDGSDLELRLKGGGSHCAGTVEVEIQKLVGKVCDR SWGLKEADVVCRQLGCGSALKTSYQVYSKTKATNTWLFVSSCNGNETSLWDCKNWQ WGGLSCDHYDEAKITCSAHRKPRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASV LCRELQCGTVVSRYTQIRLVNGKTPCEGRVELNILGSWGSLCNSHWDMEDAHVLCQQL KCGVALSIPGGAPFGKGSEQVWRHMFHCTGTEKHMGDCSVTALGASLCSSGQVASVIC SGNQSQTLSPCNSSSSDPSSSIISEESGVACIGSGQLRLVDGGGRCAGRVEVYPGASWGT I CDDSWDLNDAHVVCKQLSCGWAINATGSAHFGEGTGPIWLDEINCNGKESHIWQCHSH GWGRHNCRHKEDAGVICSEFMSLRLISENSRETCAGRLEVFYNGAWGSVGRNSMSPAT VGVVCRQLGCADRGDISPASSDKTVSRHMWVDNVQCPKGPDTLWQCPSSPWKKRLAS PSEETWITCANKIRLQEGNTNCSGRVEIWYGGSWGTVCDDSWDLEDAQVVCRQLGCGS ALEAGKEPAFGQGTGPIWLNEVKCKGNEPSLWDCPARSWGHSDCGHKEDAAVTCSEIA KSRESLHATGRSSFVALAIFGVILLACLIAFLIWTQKRRQRQRLSVFSGGENSVHQIQYR E MNSCLKADETDMLNPSGDHSEVQ (SEQ ID NO: 32) EXAMPLE 6

[0219] DELETION OF S520-C571 and THREE UNIQUE SWINE AMINO ACIDS USING A dsODN TEMPLATE

[0220] This Example provides the same deletion as that in EXAMPLE 5 with the exception that a short homology arm template contained within a plasmid is used (FIG. 9). In this example, the construct used includes:

[0221] GGGAGGCGTTCGGGCCAC GTGCTGTGCAGGGA

ACTACAGTGCGGCACTGTGGTTTCAAGTGAGACCCAGGGAATGTGTTCACTTTGTTC CC ATGCCATGAAGGCTccaCTGTGGCCCGAACGCCTCCC (SEQ ID NO: 33)

[0222] Where:

[0223] Underlined: Universal gRNA

[0224] Italic: 5’ HR ARM

[0225] Bold Italic: 3’ HR Arm

[0226] Lower case: PAM sequence

[0227] The resulting protein is the same as that in EXAMPLE 5 (SEQ ID NO: 32). EXAMPLE 7

[0228] DELETION OF S482-V496 INCLUDING TWO UNIQUE SWINE AA USING AN ssODN TEMPLATE

[0229] This Example results in a 15aa deletion or a 45bp deletion of the gene (FIG. 10). The deletion removes a so called“ligand binding domain” and 3 unique aa. The HDR template for this deletion may comprise:

[0230] 5’ TCAGCCCACAGGAAACCCAGGCTGGTTGGAGGGGACATTCCCTGCTGTGAT TCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGG 3’ (SEQ ID NO: 34)

[0231] However, it is noted that the HDR sequence may be longer or shorter in one or both of the arms than that provided herein.

[0232] The TALENs used may include: ssCD163 7.4 L/R. When CRISPR/CAS9 is used the gRNAs may include: ssCD163 g7.1, ssCD163, g7.2, ssCD163, g7.3. This strategy is illustrated in FIG.11.

[0233] The edited protein has the following sequence where the deleted portion SGRVEVQHGDTWGTV (SEQ ID NO: 35) is deleted between the cysteine (C) residues highlighted in gray below. [0234] MVLLEDSGSADFRRCSAHLSSFTFAVVAVLSACLVTSSLGGKDKELRLTGGENK CSGRVEVKVQEEWGTVCNNGWDMDVVSVVCRQLGCPTAIKATGWANFSAGSGRIWM DHVSCRGNESALWDCKHDGWGKHNCTHQQDAGVTCSDGSDLEMRLVNGGNRCLGRI EVKFQERWGTVCDDNFNINHASVVCKQLECGSAVSFSGSANFGEGSGPIWFDDLVCNG NESALWNCKHEGWGKHNCDHAEDAGVICLNGADLKLRVVDGLTECSGRLEVKFQGE WGTICDDGWDSDDAAVACKQLGCPTAVTAIGRVNASEGTGHIWLDSVSCHGHESALW QCRHHEWGKHYCNHNEDAGVTCSDGSDLELRLKGGGSHCAGTVEVEIQKLVGKVCDR SWGLKEADVVCRQLGCGSALKTSYQVYSKTKATNTWLFVSSCNGNETSLWDCKNWQ WGGLSCDHYDEAKITCSAHRKPRLVGGDIPCCDSDFSLEAASVLCRELQCGTVVSLLGG AHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCSRYTQIRLVNGK TPCEGRVELNILGSWGSLCNSHWDMEDAHVLCQQLKCGVALSIPGGAPFGKGSEQVWR HMFHCTGTEKHMGDCSVTALGASLCSSGQVASVICSGNQSQTLSPCNSSSSDPSSSIISE E SGVACIGSGQLRLVDGGGRCAGRVEVYPGASWGTICDDSWDLNDAHVVCKQLSCGWA INATGSAHFGEGTGPIWLDEINCNGKESHIWQCHSHGWGRHNCRHKEDAGVICSEFMSL RLISENSRETCAGRLEVFYNGAWGSVGRNSMSPATVGVVCRQLGCADRGDISPASSDKT VSRHMWVDNVQCPKGPDTLWQCPSSPWKKRLASPSEETWITCANKIRLQEGNTNCSGR VEIWYGGSWGTVCDDSWDLEDAQVVCRQLGCGSALEAGKEPAFGQGTGPIWLNEVKC KGNEPSLWDCPARSWGHSDCGHKEDAAVTCSEIAKSRESLHATGRSSFVALAIFGVILL ACLIAFLIWTQKRRQRQRLSVFSGGENSVHQIQYREMNSCLKADETDMLNPSGDHSEVQ

(SEQ ID NO: 36)

EXAMPLE 8

[0235] DELETION OF S482-V496 AND TWO UNIQUE AA USING AN dsODN TEMPLATE

[0236] In this Example, a dsODN template is used instead of an ssODN template (FIG.12). The template is inserted in a plasmid and liberated by coinjection of the appropriate restriction enzyme.

[0237] In this example, the construct comprises:

[0238] GGGAGGCGTTCGGGCCACAGcggTGATTTTTCAGCCCACAGGAAACCCAGGCTG GTTGGAGGGGACATTCCCTGCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGC TGTGCAGGGAAGCTccaCTGTGGCCCGAACGCCTCCC (SEQ ID NO: 37)

[0239] Where:

[0240] Underlined: Universal gRNA

[0241] Italic: 5’ HR ARM

[0242] Italic Bold: 3’ HR Arm

[0243] Lowercase: PAM sequence EXAMPLE 9

[0244] DELETION OF Q488-S531 AND FOUR UNIQUE AA USING AN ssODN TEMPLATE

[0245] This is a deletion of 44 AA and 132 bp and 4 unique AA (FIG.12). The HDR template for this deletion may comprise:

[0246] 5’ AGGCTGGTTGGAGGGGACATTCCCTGCTCTGGTCGTGTTGAAGTAGGAC AGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCACGAGTCC 3’ (SEQ ID NO: 38)

[0247] However, it is noted that the HDR sequence may be longer or shorter in one or both of the arms than that provided herein.

[0248] TALENs useful for creating this deletion include ssCD163 7.1 L/R. When CRISPR is used, gRNAs useful for making the deletion include: ssCD163 g7.6, ssCD163, g7.7

[0249] The edited protein has the following sequence where the sequence: QHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFGEGS (SEQ ID NO: 39) is deleted between the valine (V) and Glycine (G) residues highlighted in gray below:

[0250] MVLLEDSGSADFRRCSAHLSSFTFAVVAVLSACLVTSSLGGKDKELRLTGGENK CSGRVEVKVQEEWGTVCNNGWDMDVVSVVCRQLGCPTAIKATGWANFSAGSGRIWM DHVSCRGNESALWDCKHDGWGKHNCTHQQDAGVTCSDGSDLEMRLVNGGNRCLGRI EVKFQERWGTVCDDNFNINHASVVCKQLECGSAVSFSGSANFGEGSGPIWFDDLVCNG NESALWNCKHEGWGKHNCDHAEDAGVICLNGADLKLRVVDGLTECSGRLEVKFQGE WGTICDDGWDSDDAAVACKQLGCPTAVTAIGRVNASEGTGHIWLDSVSCHGHESALW QCRHHEWGKHYCNHNEDAGVTCSDGSDLELRLKGGGSHCAGTVEVEIQKLVGKVCDR SWGLKEADVVCRQLGCGSALKTSYQVYSKTKATNTWLFVSSCNGNETSLWDCKNWQ WGGLSCDHYDEAKITCSAHRKPRLVGGDIPCSGRVEVGQIWAEEFQCEGHESHLSLCPV APRPDGTCSHSRDVGVVCSRYTQIRLVNGKTPCEGRVELNILGSWGSLCNSHWDMEDA HVLCQQLKCGVALSIPGGAPFGKGSEQVWRHMFHCTGTEKHMGDCSVTALGASLCSSG QVASVICSGNQSQTLSPCNSSSSDPSSSIISEESGVACIGSGQLRLVDGGGRCAGRVEVY P GASWGTICDDSWDLNDAHVVCKQLSCGWAINATGSAHFGEGTGPIWLDEINCNGKESH IWQCHSHGWGRHNCRHKEDAGVICSEFMSLRLISENSRETCAGRLEVFYNGAWGSVGR NSMSPATVGVVCRQLGCADRGDISPASSDKTVSRHMWVDNVQCPKGPDTLWQCPSSP WKKRLASPSEETWITCANKIRLQEGNTNCSGRVEIWYGGSWGTVCDDSWDLEDAQVV CRQLGCGSALEAGKEPAFGQGTGPIWLNEVKCKGNEPSLWDCPARSWGHSDCGHKED AAVTCSEIAKSRESLHATGRSSFVALAIFGVILLACLIAFLIWTQKRRQRQRLSVFSGGE N SVHQIQYREMNSCLKADETDMLNPSGDHSEVQ (SEQ ID NO: 40) EXAMPLE 10

[0251] DELETION OF Q488-S531 AND FOUR UNIQUE AA USING A dsODN TEMPLATE

[0252] This embodiment results in the same sequence as SEQ ID NO: 40 (FIG.13). However, when the dsODN template is introduced into the cell it is contained within a plasmid. The template (insert) is liberated from the plasmid by contemporaneous introduction of CAS9 endonuclease to release the template (although, those of skill in the art will appreciate that any appropriate endonuclease or restriction enzyme could be used). In this embodiment, the construct has the following sequence:

[0253] GGGAGGCGTTCGGGCCACAGcggTGATCCCAGGCTGGTTGGAGGGGACATTCCC TGCTCTGGTCGTGTTGAAGTAGGACAGATCTGGGCTGAAGAATTCCAGTGTGAGGGGC ACGAGTCCCACGCTccaCTGTGGCCCGAACGCCTCCC (SEQ ID NO: 41)

[0254] Wherein:

[0255] Underlined: Universal gRNA

[0256] Italic: 5’ HR ARM

[0257] Italic Bold: 3’ HR Arm

[0258] Lower case: PAM sequence EXAMPLE 11

[0259] DELETION OF Q488, T495, L521, S531 and/or D558 USING AN ssODN TEMPLATE

[0260] Q488, T495, L521, S531 and/or D558 are deleted using either multiplex gene editing or a single template. In this example, all five of the amino acids unique to swine may be deleted without otherwise altering the CD163 protein. In other embodiments, only a subset of the unique residues are deleted.

[0261] In various embodiments the ssODN template may have a sequence comprising

[0262] AATCGGCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCC CTTTCTCACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCAT ATTCTCCCTCACCGAAATGCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGA GGGGACATTCCCTGCTCTGGTCGTGTTGAAGTACATGGAGACACGTGGGGCGTCTGT GATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTACAGTGCGGC ACTGTGGTTTCCCTGGGGGGAGCTCACTTTGGAGAAGGAGGACAGATCTGGGCTGA AGAATTCCAGTGTGAGGGGCACGAGTCCCACCTTTCACTCTGCCCAGTAGCACCCCG CCCTGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGTCTGCTCAAGTGAGACCC AGGGAATGTGTTCACTTTGTTCCCATGCCATGAAGAGGGTAGGGTTAGGTAGTCACA GACATCTTTTTAAAGCCCTGTCTCCTTCCAGGATACACACAAATCCGCTTGGTGAAT GGCAAGACCCCATGTGAAGGAAGAGTGGAGCTCA, with homology arms of approximately 200 bp. (SEQ ID NO: 46)

[0263] TALENs useful to make this edit include ssCD163 7.1 L/R, ssCD1637.5 L/R, ssCD163 7.6 L/R, ssCD1637.7 L/R. When CRISPR/CAS9 is used, guide RNA includes gRNAs: ssCD163 g7.4, ssCD163 g7.5, ssCD163 g7.6, ssCD163 g7.7. EXAMPLE 12

[0264] CONVERSION OF INTRON 6 - 3’ SPLICE ACCEPTOR TO A NON-FUNCTIONAL MOTIF

[0265] Splicing out of introns during transcription is facilitated by the presence of a splice donor at the start of an intron (5’) and a splice acceptor at the end of the intron (3’). The splice donor has the consensus GT while the splice acceptor has the consensus AG. Small nuclear ribonucleoproteins (snRP’s) search for the splice acceptor and slice donor during transcription and join together the exons to provide the intron-less mRNA. If the splice donor or splice acceptor is mutated so as not to comprise the consensus, either the beginning of the intron or the end of the intron is not recognized. In this example, the splice acceptor is mutated so as not to comprise AG.

[0266] By changing the splice acceptor, the beginning of the intron will be recognized but the snRPs will not recognize the end of the intron and will instead pass on to the next splice acceptor. In this case, the next splice acceptor will be that at the end of intron 7 (just prior to exon 8). The result will be that the entire exon 7 will be spliced out of the mRNA with exon 6 being ligated directly to exon 8, thus also leaving the entire sequence of exon 7 intact within the chromosome. For this embodiment, base editing can be used. Specifically, the adenine (A) residue can be converted to inosine, by using an adenine base editor (ABE) as described herein. The nick caused by the ABE in the second strand will lead to repair by the cell to base pair the inosine with a cytosine (C). This base change will render the intron 6 splice acceptor unreadable resulting in the small nuclear ribonucleoproteins (snRPs) recognizing the 7 ^th intron splice acceptor as the pair of the 6 ^th intron splice donor and resulting in the excision of the 6 ^th intron, 7 ^th exon and 7 ^th intron during mRNA transcription. Upon replication of the genome, the inosine will be read as a guanine (G). The following gRNAs can be utilized to make the base changes: ssCD163 g7.8, ssCD163 g7.9 and ssCD163 g7.10.

[0267] To prevent the translation of the SRCR5 trans membrane domain of CD163, a known binding region of PRRSV, the splice acceptor region of intron 6 of CD163, flanking exon 7 which contains the gene sequence of the SCRC5 trans membrane domain, was modified in the genome of porcine fibroblasts. Figure 14A shows a schematic of the genomic sequence of the splice acceptor region of intron 6/exon 7 junction of the porcine CD163 gene, 5’- GCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGAGGGG-3’ (SEQ ID NO: 87). The splice acceptor site is highlighted with the nucleotide sequence of AG. An Adenine Base Editor (ABE) bound to an sgRNA targeting the intron 6/exon7 junction, ssCD163 g7.10, 5’- TTTCAGCCCACAGGAAACCC-3’ (SEQ ID NO: 88), was used to deaminate the Adenine to an Inosine, converting the AG to IG. Upon replication either of the cell or by amplification in PCR, the IG is then converted to GG, as can be seen at bases 12 and 13 of the original sequenced region GCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGAGGGG (SEQ ID NO: 86) as compared to bases 12 and 13 of the sequenced region after purification from the modified cell GCTATTTTTTCGGCCCACAGGAAACCCAGGCTGGTTGGAGGGG (SEQ ID NO: 89).

[0268] To test the editing efficiency of the Adenine Base Editor after transfection into the cell as RNA, 600,000 porcine fibroblast cells were transfected with 10µg of sgRNA ssCD163 g7.10 along with 10 µg Adenine Base Editor RNA (codon optimized). The cells were incubated for three days at 30°C after transfection prior to lysis and PCR amplification with primers that flank the junction site. Amplicons were sequenced by next generation sequencing (NGS) and the top four variants are show in FIG. 14B along with the relative percentages. Presence of the modification was confirmed by PCR to match SEQ ID NOs: 89-91.

[0269] To test the effect of transfecting the Adenine Base Editor as a protein in complex with guide RNA ssCD163 g7.10, 600,000 porcine fibroblast cells were transfected with 10µg ssCD163 g7.10 (guide RNA) along with 75µg ABE 7.10 protein to form ABE ribonuclease particles (ABE RNP). Cells were incubated 3 days at 37°C after transfection prior to lysis and PCR amplification with primers that flank the junction site. Amplicons were sequenced by NGS and the top 4 variants are shown in FIG. 14C along with relative percentages. Presence of the modification was confirmed by PCR to match SEQ ID NOs: 89-91.

[0270] To test the effect of incubation at a lower temperature post transfection on editing efficiency, 600,000 porcine fibroblast cells were transfected with 10µg ssCD163 g7.10 (guide RNA) along with 75µg ABE 7.10 protein to form ABE ribonuclease particles (ABE RNP). Cells were incubated 3 days at 30°C after transfection prior to lysis and PCR amplification with primers that flank the junction site. Amplicons were sequenced by NGS and the top 3 variants are shown in FIG.14D along with relative percentages. Presence of the modification was confirmed by PCR to match SEQ ID NOs: 89-91. EXAMPLE 13

[0271] CONVERSION OF Q488, T495, L521, S531 and/or D558 TO A SPECIALIZED AMINO ACID USING AN ssODN TEMPLATE

[0272] In this example, amino acid residues of CD163 that are unique to swine SRCR5 are converted to specialized amino acids. For example, the residues may be converted to a particular amino acid having a desired quality. Such qualities may include a desire for a non-polar or neutral amino acid, a polar and neutral amino acid, an acidic and polar amino acid or a basic and polar amino acid. In addition, unique amino acids may be converted to branched chain aa’s. In yet other embodiments, the unique aa may be converted to an aa of a similar size such as, for example glycine.

[0273] Any one of the following underlined codons may be altered.

[0274] AATCGGCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCC CTTTCTCACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCAT ATTCTCCCTCACCGAAATGCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGA GGGGACATTCCCTGCTCTGGTCGTGTTGAAGTAcaaCATGGAGACACGTGGGGCaccG TCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTACAGT GCGGCACTGTGGTTTCCctcCTGGGGGGAGCTCACTTTGGAGAAGGAagtGGACAGAT CTGGGCTGAAGAATTCCAGTGTGAGGGGCACGAGTCCCACCTTTCACTCTGCCCAGT AGCACCCCGCCCTgacGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGTCTGCTC AAGTGAGACCCAGGGAATGTGTTCACTTTGTTCCCATGCCATGAAGAGGGTAGGGTT AGGTAGTCACAGACATCTTTTTAAAGCCCTGTCTCCTTCCAGGATACACACAAATCC GCTTGGTGAATGGCAAGACCCCATGTGAAGGAAGAGTGGAGCTCA. (SEQ ID NO: 86)

[0275] In one embodiment, the ssODN template may have the sequence:

[0276] AATCGGCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCC CTTTCTCACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCAT ATTCTCCCTCACCGAAATGCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGA GGGGACATTCCCTGCTCTGGTCGTGTTGAAGTAGGGCATGGAGACACGTGGGGCGG GGTCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTACA GTGCGGCACTGTGGTTTCCGGGCTGGGGGGAGCTCACTTTGGAGAAGGAGGGGGA CAGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCACGAGTCCCACCTTTCACTCTGC CCAGTAGCACCCCGCCCTGGGGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGT CTGCTCAAGTGAGACCCAGGGAATGTGTTCACTTTGTTCCCATGCCATGAAGAGGGT AGGGTTAGGTAGTCACAGACATCTTTTTAAAGCCCTGTCTCCTTCCAGGATACACAC AAATCCGCTTGGTGAATGGCAAGACCCCATGTGAAGGAAGAGTGGAGCTCA. (SEQ ID NO: 85)

EXAMPLE 14

[0277] CONVERSION OF Q488, T495, L521, S531 and/or D558 TO A SPECIALIZED AMINO ACID WITHOUT USING AN ssODN TEMPLATE

[0278] In this example, amino acid residues of CD163 that are unique to swine SCRC5 are converted to specialized amino acids. For example, the residues may be converted to a particular amino acid having a desired quality. Such qualities may include a desire for a non-polar or neutral amino acid, a polar and neutral amino acid, an acidic and polar amino acid or a basic and polar amino acid. In addition, unique amino acids may be converted to branched chain aa’s. In yet other embodiments, the unique aa may be converted to an aa of a similar size such as, for example, glycine. Changed bases are underlined. TALENs useful to make this edit include ssCD163 7.1 L/R, ssCD1637.5 L/R, ssCD1637.6 L/R, ssCD1637.7 L/R. CRISPR/Cas9 may also be used to make any of the above edits.

[0279] Individual base changes to one or more of the underlined codons may be made using an appropriate base editor (ABE or CBE) without an ssODN template.

[0280] AATCGGCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCC CTTTCTCACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCAT ATTCTCCCTCACCGAAATGCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGA GGGGACATTCCCTGCTCTGGTCGTGTTGAAGTACAACATGGAGACACGTGGGGCAC CGTCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTACA GTGCGGCACTGTGGTTTCCCTCCTGGGGGGAGCTCACTTTGGAGAAGGAAGTGGAC AGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCACGAGTCCCACCTTTCACTCTGCC CAGTAGCACCCCGCCCTGACGGGACATGTAGCCACAGCAGGGACGTCGGCGTAGTC TGCTCAAGTGAGACCCAGGGAATGTGTTCACTTTGTTCCCATGCCATGAAGAGGGTA GGGTTAGGTAGTCACAGACATCTTTTTAAAGCCCTGTCTCCTTCCAGGATACACACA AATCCGCTTGGTGAATGGCAAGACCCCATGTGAAGGAAGAGTGGAGCTCA. (SEQ ID NO: 86)

[0281] The following base changes may be made using an ABE or CBE base editor with a gRNA targeting any of the aforementioned codons in order to change the corresponding amino acid within CD163 as described herein:

EXAMPLE 15

[0282] DISRUPTION OF THE SPLICE ACCEPTOR BETWEEN INTRON 6 AND EXON 7 OF CD163 USING CRISPR/Cas9 AND AN HDR TEMPLATE

[0283] Splicing out of introns during transcription is facilitated by the presence of a splice donor at the start of an intron (5’) and a splice acceptor at the end of the intron (3’). The splice donor has the consensus GT while the splice acceptor has the consensus AG. Small nuclear ribonucleoproteins (snRP’s) search for the splice acceptor and slice donor during transcription and join together the exons to provide the intron-less mRNA. If the splice donor or splice acceptor is mutated so as not to comprise the consensus, either the beginning of the intron or the end of the intron is not recognized. In this example, the splice acceptor is mutated so as not to comprise AG.

[0284] By changing the splice acceptor, the beginning of the intron will be recognized but the snRPs will not recognize the end of the intron and will instead pass on to the next splice acceptor. In this case, the next splice acceptor will be that at the end of intron 7 (just prior to exon 8). The result will be that the entire exon 7 will be spliced out of the mRNA with exon 6 being ligated directly to exon 8, thus also leaving the entire sequence of exon 7 intact within the chromosome.

[0285] Disruption of the splice acceptor can be achieved using homology dependent repair in pig fibroblasts. 600,000 porcine fibroblast cells from two separate lines (FIG.15B, lines 2 and 3) were transfected with 120pmol ssCD163 g7.8 crRNA/tracrRNA duplex (IDT) complexed with 17.2 µg Alt-R S.p. HiFi Cas9 Nuclease (IDT) and 0.1 nanomoles of HDR template

(TATTGGAATCATATTCTCCCTCACCGAAATGCTATTTTTTCGGCTCACCGGAAACC C AGGCTGGTTGGAGGGGACATTCCCTGCTCTGGT (SEQ ID NO: 94)). Cells were incubated 2 days at 30°C or 37°C after transfection prior to analysis by PCR amplification with primers (ssCD163 SD F1, ssCD163 SD R1) that flank the cut site and Restriction Fragment Length Polymorphism (RFLP) assay using the novel Msp1. Single cell derived colonies were produced and analyzed by the same RFLP assay. As can be seen in FIG.15C, clones 49, 51, 61, and 65 have banding consistent with homozygous HDR with to match the template, while clones 53, 54, 55, 56, 57, 62, 63, 64, 67, and 69 have banding consistent with heterozygous HDR.

Clone 51 was selected for Sanger sequencing of the amplicon revealing the expected

homozygous HDR of the target nucleotides, as can be seen in FIG.15D. [0286] FIG.15A is a schematic of the intron 6/ exon 7 junction of porcine CD163 gene. The splice acceptor site is noted with the nucleotide sequence of AG. Below is the 43-bp of an homology dependent repair template (HDR) used to disrupt the slice acceptor by converting AG => GG (altered G is underlined). Two additional silent SNPs introduced into the template 3’ of the slice acceptor (underlined) were used to alter the PAM sequence required for Cas9 binding and creation of a novel Msp1 restriction site, respectively. The SNP in PAM domain would inhibit binding and cutting of the HDR allele when using gRNA ssCD163 g7.8. [0287] Primers for RFLP [0288] ssCD163 SD F1: 5’-GGTGTGCCTTTGACTCCAGA-3’(SEQ ID NO: 97) [0289] ssCD163 SD R1: 5’-TGCCCCTCACACTGGAATTC-3’(SEQ ID NO: 98) [0290] Amplicon without HDR: [0291] GGTGTGCCTTTGACTCCAGATTACAGTAAATGGAGGACTGAGTATAGGGCTA AAAAGTAGAGAGAATGGATGCATATTATCTGTGGTCTCCAATGTGATGAATGAA GTAGGCAAATACTCAAAGGAAAGAGAAAGCATGCTCCAAGAATTATGGGTTCCA GAAGGCAAAGTCCCAGAATTGTCTCCAGGGAAGGACAGGGAGGTCTAGAATCG GCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCCCTTTCTC ACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCATATTCT CCCTCACCGAAATGCTATTTTTTCAGCCCACAGGAAACCCAGGCTGGTTGGAGG GGACATTCCCTGCTCTGGTCGTGTTGAAGTACAACATGGAGACACGTGGGGCAC CGTCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTA CAGTGCGGCACTGTGGTTTCCCTCCTGGGGGGAGCTCACTTTGGAGAAGGAAGT GGACAGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCA (SEQ ID NO: 95) [0292] Amplicon with HDR: [0293] GGTGTGCCTTTGACTCCAGATTACAGTAAATGGAGGACTGAGTATAGGGCTA AAAAGTAGAGAGAATGGATGCATATTATCTGTGGTCTCCAATGTGATGAATGAA GTAGGCAAATACTCAAAGGAAAGAGAAAGCATGCTCCAAGAATTATGGGTTCCA GAAGGCAAAGTCCCAGAATTGTCTCCAGGGAAGGACAGGGAGGTCTAGAATCG GCTAAGCCCACTGTAGGCAGAAAAACCAAGAGGCATGAATGGCTTCCCTTTCTC ACTTTTCACTCTCTGGCTTACTCCTATCATGAAGGAAAATATTGGAATCATATTCT CCCTCACCGAAATGCTATTTTTTCGGCTCACCGGAAACCCAGGCTGGTTGGAGGG GACATTCCCTGCTCTGGTCGTGTTGAAGTACAACATGGAGACACGTGGGGCACC GTCTGTGATTCTGACTTCTCTCTGGAGGCGGCCAGCGTGCTGTGCAGGGAACTAC AGTGCGGCACTGTGGTTTCCCTCCTGGGGGGAGCTCACTTTGGAGAAGGAAGTG GACAGATCTGGGCTGAAGAATTCCAGTGTGAGGGGCA (SEQ ID NO: 96) EXAMPLE 16

[0294] PRIME EDITING OF ssCD163 EXON 7 SPLICE ACCEPTOR (SA) TO BLOCK mRNA SPLICING

[0295] Splicing out of introns during transcription is facilitated by the presence of a splice donor at the start of an intron (5’) and a splice acceptor at the end of the intron (3’). The splice donor has the consensus GT while the splice acceptor has the consensus AG. Small nuclear ribonucleoproteins (snRP’s) search for the splice acceptor and slice donor during transcription and join together the exons to provide the intron-less mRNA. If the splice donor or splice acceptor is mutated so as not to comprise the consensus, either the beginning of the intron or the end of the intron is not recognized. In this example, the splice acceptor is mutated so as not to comprise AG.

[0296] By changing the splice acceptor, the beginning of the intron will be recognized but the snRPs will not recognize the end of the intron and will instead pass on to the next splice acceptor. In this case, the next splice acceptor will be that at the end of intron 7 (just prior to exon 8). The result will be that the entire exon 7 will be spliced out of the mRNA with exon 6 being ligated directly to exon 8, thus also leaving the entire sequence of exon 7 intact within the chromosome.

[0297] For this embodiment, prime editing can be used. Specifically, the bases at the splice acceptor of Exon 7, AG, as seen in FIG. 16C, can be converted to GG or AA by using a prime editor as described herein. This change will render the intron 6 splice acceptor unreadable resulting in the small nuclear ribonucleoproteins (snRPs) recognizing the 7 ^th intron splice acceptor as the pair of the 6 ^th intron splice donor and resulting in the excision of the 6 ^th intron, 7 ^th exon and 7 ^th intron during mRNA transcription. The following pegRNAs can be utilized to make modify the splice acceptor (SA): ssCD163 ex7 SA pegRNA-1 or ssCD163 ex7 SA pegRNA-2.

[0298] To prevent the translation of the SRCR5 trans membrane domain of CD163, a known binding region of PRRSV, the splice acceptor region of intron 6 of CD163, flanking exon 7 which contains the gene sequence of the SCRC5 trans membrane domain, is modified in the genome of porcine cells. Figure 16A shows a schematic of the modification of the splice acceptor region of intron 6/exon 7 junction of the porcine CD163 gene using a prime editor in complex with a pegRNA. [0299] FIG.16B shows the splice acceptor site, highlighted with the nucleotide sequence of AG. A Prime Editor bound to a pegRNA targeting the intron 6/exon7 junction, ssCD163 ex7 SA pegRNA-1, (SEQ ID NO: 92), or ssCD163 ex7 SA pegRNA-2 (SEQ ID NO: 93), is used to replace the AG to XX which can be GG or AA.

[0300] To test the editing efficiency of the Prime Editor after transfection into the cell, 600,000 porcine fibroblast cells were transfected with pegRNA along with the prime editor as either a protein complex or as codon optimized RNA. The cells are incubated for three days at 30°C or 37°C after transfection prior to lysis and PCR amplification with primers that flank the junction site. Amplicons are sequenced by next generation sequencing (NGS).

[0301] The following paragraphs enumerated consecutively from 1 through 19 provide for various additional aspects of the present invention. In one embodiment, in a first paragraph: All patents, publications, and journal articles set forth herein are hereby incorporated by reference herein; in case of conflict, the instant specification is controlling.

1. A genetically edited porcine animal being resistant to porcine reproductive and respiratory syndrome virus (PRRSV) comprising a genetic modification which alters the expression or activity of CD 163 and wherein the gene retains a majority of exon 7 of the CD163 gene.

2. A genetically edited porcine animal according to paragraph 1 wherein the CD163 gene has been edited to eliminate the ability of CD163 to bind to porcine reproductive and respiratory syndrome virus (PRRSV).

3. The genetically edited porcine animal according to paragraphs 1-2, wherein the genetic edit comprises in-frame deletions of portions exon 7 of the CD163 gene encoding amino acid residues unique to swine in the CD 163 protein

4. A genetically edited porcine animal according to paragraphs 1-3 wherein edits to the CD163 gene comprise in-frame deletions of Q488, T495, L521, S531, D558 and combinations thereof.

5. The genetically edited porcine animal of paragraphs 1-4, wherein the deletions range in size from, 1 aa to 52 aa and combinations thereof.

6. The genetically edited porcine animal of paragraphs 1-5, wherein the edits to the gene range in size from 3 to 6 to 156 basepairs.

7. The genetically edited porcine animal according to paragraphs 1-6, wherein the genetic modification comprises one or more conversions comprising Q488K, T495S, L521I, S531N and D558E.

8. The genetically edited porcine animal according to paragraphs 1-7, wherein the splice acceptor site at the end of intron 6 is edited so that the site is not recognized. 9. The genetically edited porcine animal according to paragraphs 1-8, wherein exon 7 is not transcribed into mRNA.

10. The genetically edited porcine animal of paragraph 9, wherein a base editor is used.

11. The genetically edited porcine animal of paragraph 10, wherein the AG of the splice acceptor is converted to AA.

12. A method to provide porcine animals that are resistant to PRRSV comprising: editing the CD163 gene of the animal to convert or delete Q488, T495, L 521, S531, D558 and combinations thereof.

13. The method of paragraph 12, wherein the gene edits result in deletions of discrete regions of scavenger receptor cysteine-rich (SRCR) domain 5.

14. The method of paragraphs 12-13, wherein the portions of the CD163 gene deleted comprise in-frame deletions comprising unique swine residues and leaving a majority of the gene and the protein intact.

15. The method of paragraphs 12-14, wherein the deletions range in size from, range in size from, 22 aa to 52 aa and combinations thereof.

16. The method of paragraphs 12-15, wherein the edits to the gene range in size from 3 to 6 to 156 basepairs and combinations thereof.

17. The method of paragraphs 12-16, wherein the edited gene is translated into a functional protein.

18. The method of paragraphs 12-17, wherein Q488, T495, L521, S531 and D558 are converted to K, S, I, N and E respectively.

19. Any invention described herein.

[0302] While this invention has been described in conjunction with the various exemplary embodiments outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent to those having at least ordinary skill in the art. Accordingly, the exemplary embodiments according to this invention, as set forth above, are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention. Therefore, the invention is intended to embrace all known or later-developed alternatives, modifications, variations, improvements, and/or substantial equivalents of these exemplary embodiments.