Genetically-Edited Swine

Abstract

The present invention relates to genetically-edited swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene. In particular it relates to genetically-edited swine comprising an introgressed warthog allele in the RALA gene of domestic pigs. The invention also related to methods of producing such swine, and cells derived from swine having such introgressed sequences.

Claims

1. A genetically-edited swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene.

2. The genetically-edited swine of claim 1 wherein the swine is a domestic pig.

3. The genetically-edited swine of claim 1 wherein the introgressed heterologous nucleic acid sequence comprises a heterologous RELA allele.

4. The genetically-edited swine of claim 3 wherein the introgressed heterologous sequence comprises a trans-species heterologous RELA allele.

5. The genetically-edited swine of claim 1 wherein the introgressed heterologous nucleic acid sequence converts a wild-type RELA allele to the corresponding warthog RELA allele.

6. The genetically-edited swine of claim 1 which comprises an introgressed RELA allele that differs from the wild-type RELA allele sequence by changes in two or more bases.

7. The genetically-edited swine of claim 1 which comprises an introgressed heterologous nucleic acid which is 50 or more bases in length.

8. The genetically-edited swine of claim 1 wherein all cells of the genetically-edited swine contain the introgressed heterologous nucleic acid sequence.

9. The genetically-edited swine of claim 1 wherein the introgressed heterologous nucleic acid sequence changes the sequence of the RELA protein.

10. The genetically-edited swine of claim 1 wherein the introgressed heterologous nucleic acid sequence changes sequences in the region of the RELA gene which encodes the transactivation domain of RELA.

11. The genetically-edited swine of claim 10 wherein the introgressed heterologous nucleic acid sequence changes the region of the RELA gene which encodes amino acids 448 to 531 of RELA.

12. The genetically-edited swine of claim 1 which comprises an introgressed trans-species allele of the RELA gene.

13. The genetically-edited swine of claim 1 which is a domestic pig comprising an introgressed warthog RELA gene.

14. The genetically-edited swine of claim 1 wherein the swine is bi-allelic for an introgressed RELA allele.

15. The genetically-edited swine of claim 1 which is a domestic pig and wherein the introgressed heterologous nucleic acid sequence causes a change in one or more of the following amino acids of RELA: T448; S485; and S531.

16. The genetically-edited swine of claim 15 wherein all three amino acids are changed.

17. The genetically-edited swine of claim 15 comprising at least one of the following amino acid changes to RELA: T448A, S485P, and S531P.

18. The genetically-edited swine of claim 1 which is a domestic pig that comprises an introgressed heterologous nucleic acid sequence which results in the following amino acid changes to RELA: T448A, S485P and S531P.

19. The genetically-edited swine of claim 18 in which no amino acid changes other than T448A, S485P and S531P of RELA are caused by the introgressed heterologous nucleic acid.

20. The genetically-edited swine of claim 1 which is a domestic pig wherein the RELA gene has been edited such that it encodes the sequence as set out below: TABLE-US-00007 (SEQIDNO17) LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHT AEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIA DM.

21. The genetically-edited swine of claim 1 which is a domestic pig that has improved tolerance to ASFV infection resulting from the introgressed heterologous nucleic acid sequence.

22. A cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell of a swine comprising an introgressed heterologous nucleic acid sequence in the RELA gene.

23. A cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell of a swine according to claim 22 which comprises an introgressed warthog RELA allele.

24. A method of producing a genetically-edited swine having an introgressed heterologous nucleic acid sequence in the RELA gene, the method comprising the steps of: providing a swine zygote; introducing into said zygote a site-specific nuclease, the nuclease being adapted to target a desired genomic sequence in the RELA gene to be edited, and to introduce a double stranded break; introducing a template nucleic acid comprising the heterologous nucleic acid sequence to be introgressed into the RELA gene, the heterologous sequence being flanked by sequences homologous to genomic RELA sequences; incubating said zygote under suitable conditions to permit cutting of the genome by the site-specific nuclease and introgression of the heterologous nucleic acid sequence into the RELA gene by homology directed repair; and generating an animal from said zygote.

25. The method of claim 24 wherein the swine is a domestic pig.

26. The method of claim 24 wherein the site-specific nuclease is adapted to target and cut within the region of the RELA gene encoding the transactivation domain of RELA.

27. The method of claim 24 wherein the site-specific nuclease is adapted to target and cut within exon 9 of the RELA gene.

28. The method of claim 27 wherein the site-specific nuclease is adapted to target and cut upstream of the region of the RELA gene encoding amino acid T448.

29. The method of claim 24 wherein the site-specific nuclease is adapted to target and cut a sequence of the RELA gene lying between bases 1200 and 1341 with reference to SEQ ID NO 15.

30. The method of claim 24 wherein the site-specific nuclease comprises a pair of cooperating site-specific nucleases and the target site of one of the pair is GATACTGATGAGGAC (SEQ ID NO 18) and the target site of the other of the pair is CTCCGGGACGACGTC (SEQ ID NO 19).

31. The method of claim 24 wherein mRNA encoding the nuclease is introduced into the zygote.

32. The method of claim 24 wherein introgression of the heterologous nucleic acid sequence is completed in the zygote at the single cell stage.

33. The method of claim 24 in which the template nucleic acid comprises a region including the heterologous nucleic acid sequence flanked on each side by homologous sequences.

34. The method of claim 24 in which the template construct comprises a heterologous nucleic acid that is 50 or more bases in length.

35. The method of claim 24 in which the template nucleic acid comprises two or more base changes compared with the corresponding genomic nucleic acid sequence at the target site for the SSN site-specific nuclease.

36. The method of claim 24 in which the template nucleic acid comprises a region including the warthog RELA haplotype flanked on each side by homologous sequences.

37. The method of claim 24 which comprises introgressing a heterologous nucleic acid sequence that results in the following amino acid changes to RELA: T448A, S485P and S531P.

38. The method of claim 24 in which the template nucleic acid comprises a region including the warthog RELA of 251 or more nucleotides in length, which comprises a nucleic acid sequence encoding the protein sequence: TABLE-US-00008 (SEQIDNO23) ADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEY PEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPflankedby homologousregions.

39. The method of claim 24 in which the template nucleic acid is double stranded.

40. The method of claim 24 in which the template nucleic acid is provided in a plasmid.

41. The method of claim 24 in which the template is plasmid comprising a 251 bp region containing the warthog RELA haplotype flanked by homology arms of 626 bp and 799 bp.

42. The method of claim 24 comprising the steps of: providing a domestic pig zygote; introducing into said zygote a pair of cooperating site-specific nucleases, the nucleases being adapted to target the RELA gene in the region of within 20 bp of the sequence encoding T448A and introduce a double stranded break; introducing a template nucleic acid comprising a heterologous nucleic acid comprising a sequence encoding the corresponding warthog RELA haplotype flanked by sequences homologous to the genomic RELA sequence of the pig; incubating said zygote under suitable conditions to permit cutting of the genome by the site-specific nuclease and introgression of the heterologous nucleic acid by homology-directed repair; and generating a pig from said zygote.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0075] FIG. 1Design strategy for creation of a DSB and subsequent HDR of the domestic pig RELA. (a) Depiction of the RELA gene with exons as solid bars. An expanded view of a region of the final exon highlights the 3 amino acid differences between domestic pig (red) and warthog (green). Scissors indicate the intended site of the DSB. (b) DNA sequence, with solid bars representing ZFN binding sites on the domestic pig sequence. Lower panel indicates sequence changes that would concurrently change the domestic pig threonine to the warthog alanine whilst at the same time prevent re-cutting by the ZFN. (c) Design of HDR template, with 626 bp and 799 bp homology arms flanking a 251 bp region, including 5 base changes to convert the domestic pig sequence to the warthog haplotype.

[0076] FIG. 2Sequence analysis of live born piglets. The sequence of both the domestic pig and warthog encoding the three observed amino acid differences is shown above, with sequence traces from individual animals below. Inset pictures of chromosomes indicate the allelic makeup at each position in each animal (domestic pig allelered; warthog allelegreen).

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0077] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as a, an and the are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. The term swine, or variants thereof, as used herein refers to any of the animals in the Suidae family of even-toed ungulates including animals in the genus Sus and other related species, including the peccary, the babirusa, and the warthog.

[0078] The term pig or variants thereof as used herein refers to any of the animals in the genus Sus. It includes the domestic pig (Sus scrofa domesticus or Sus domesticus) and its ancestor, the common Eurasian wild boar (Sus scrofa). For the present purposes the domestic pig is considered to be a sub-species of the species Sus scrofa. It does not include the peccary, the babirusa, and the warthog.

[0079] The term domestic pig, or variants thereof, as used herein refers to an animal of the sub-species Sus scrofa domesticus.

[0080] The term RELA gene, or variants thereof, as used herein refers to the RELA (V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A gene, also known as the p65 gene, NCBI Gene ID: 100135665) gene, and includes both coding and non-coding regions, and also associated regulators promoter and enhancer regions. In preferred embodiments of the invention introgression modifies the sequence within the RELA gene ORF, and more preferably within at least one exon.

[0081] The term site-specific nuclease, or variants thereof, as used herein refers to engineered nucleases which can be configured to cut DNA at a desired location. Such site-specific nucleases are also known as engineered nucleases, targetable nucleases, genome editing nucleases, molecular scissors, and suchlike. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system (CRISPR), and meganucleases, such as hybrid meganucleases.

[0082] The term heterologous allele, or variants thereof, as used herein refers to an allele which is not present in the relevant animal. The heterologous allele can be naturally occurring in another species or genus, or it can be non-natural in any species (i.e. entirely artificial). Preferably the allele is naturally occurring in another species.

[0083] The term trans-species heterologous allele, or variants thereof, as used herein refers to an allele which does not naturally occur in the species of the relevant animal, but which occurs naturally in another species. The heterologous allele can be naturally occurring in another species, which species may be from the same or a different genus. Thus a trans-species allele is still a natural allele in the sense that it is not artificial and is found in nature, but it is introgressed to a new species to form a new animal with desired properties.

[0084] The term trans-genus heterologous allele, or variants thereof, as used herein refers to an allele which does not naturally occur in the genus of the relevant animal, but which occurs naturally in another genus. The set trans-genus heterologous alleles is thus a subset of trans-species heterologous alleles, i.e. wherein a trans-genus heterologous allele comes from outside of the relevant animal's genus, and not merely from outside of the relevant animal's species. For example, the RELA allele from warthogs is a trans-genus heterologous allele for animals in the genus Sus, and in particular to domestic pigs.

[0085] The term haplotype, or variants thereof, as used herein refers to a linked set of DNA sequence variations (typically single-nucleotide polymorphisms (SNPs)) at a specific locus on a single chromatid of a chromosome pair. In the present invention a haplotype is typically a plurality of SNPs differing between one species or genus and other, which contribute to or define a heterologous allele as between the one species or genus and the other. For example, in the case of the RELA allele in the present example there are 3 amino acid changes as between domestic pigs and warthog; these changes represent the haplotype of the heterologous RELA allele as between domestic pigs and warthogs.

[0086] The term introgression, or variants thereof, as used herein refers to the introduction of a heterologous nucleic acid sequence, especially a gene or allele, from a given source into an animal, typically by rewriting or converting an existing genomic sequence. Re-writing or converting in the present invention is achieved by HDR. The source of the heterologous nucleic acid sequence can be an animal from another species or genus, or it can be an artificial sequence.

[0087] The term allele introgression, or variants thereof, as used herein refers to a genetic edit which introduces an allele to the genome of an animal. The allele introgression can be an allele conversion or allele replacement, or it may, for example, introduce a new gene in its entirety. In preferred embodiments of the present invention the allele introgression is an allele conversion or allele replacement.

[0088] The term allele conversion or allele replacement, or variants thereof, as used herein refers to an introgression which replaces a normal, usually wild-type, allele with a heterologous allele. Conversion or replacement of a wild-type allele to a heterologous allele can in some cases involve alteration of the wild-type genomic DNA sequence to exactly match the DNA sequence of the heterologous allele from another animal. However, in other cases conversion or replacement may only require modification to the wild-type genomic DNA sequence such that the encoded protein matches the protein encoded by the heterologous allele (i.e. synonymous substitutions need not be made to the wild-type genomic sequence). The type of alteration required will depend on the manner in which the allele exerts its phenotype, e.g. via the activity of encoded protein versus regulation of transcription; in the former the encoded protein sequence is of primary importance, whereas in the latter the DNA sequence is of primary importance.

[0089] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0090] Pioneering work by the Jasin laboratory demonstrated that a single DSB in mammalian cells can lead to the transfer of a panel of single nucleotide polymorphisms (SNPs) forming an uninterrupted, short (<200 bp) haplotype to the chromosome from an extrachromosomal repair template.sup.7.

[0091] The present inventors sought to use genome editing to introduce the entire warthog RELA haplotype (which spans 251 base pairs, bearing five SNPs resulting in 3 amino acid changes) via a single nuclease-induced DSB. To the best of our knowledge, such editing-driven haplotype introgression had not been previously reported in live born animals of any mammalian species. We now demonstrate that this can be achieved efficiently in domestic pig by direct injection into the zygote cytoplasm. Strikingly, we observe single-step bi-allelic haplotype transfer by genotyping of piglets. Detailed materials and methods are provided in a separate section below, and the outline methodology, results and conclusions will now be discussed.

[0092] ZFNs can be engineered to induce a DSB at any genomic position. In this specific case, nuclease design considerations were informed by the need to transfer an entire 251 bp haplotype bearing multiple SNPs. The inventors conceived a strategy (FIG. 1a) in which a ZFN is engineered for a region immediately upstream of the haplotype-marked stretch, with the intention that single-sided invasion of the repair template by the upstream chromosome arm would then lead to a synthesis-dependent strand annealing-based transfer of the entire downstream haplotype to the endogenous locus.

[0093] A ZFN heterodimer (see Table 1 for details) was produced that binds to the region flanking 1330 to 1338 bp relative to the translational start site in the porcine RELA cDNA sequence (NM_001114281). We compared two formats of an expression construct for the ZFNs: two plasmids, each encoding a single ZFN monomer, and one plasmid that encodes both ZFN monomers spanned by a ribosome stuttering signal or a 2A peptide.sup.8, 9. We transfected these plasmids into a transformed cell line (PK15) established from the domestic pig, and compared genome editing efficiencies via the Surveyor/Cel-1 endonuclease assay.sup.10. We observed comparable on-target editing driven by either expression construct configuration. The editing efficiency driven by the RELA-directed ZFNs nearly doubled that seen with ZFNs successfully used to obtain live pigs bearing a disruption of the GGTA gene.sup.11, suggesting that these nucleases may be well-suited for in-embryo editing.

[0094] While the present inventors used ZFNs to create a DSB in the genome, such a cut can be achieved using the various other site-specific nucleases now well-known to the skilled person. For example, a suitable TALEN pair could readily be designed to target the same locus, and the CRISPR/Cas system could also be used by providing suitable guide RNA sequences to guide either wild-type or paired nickase Cas nuclease(s). Accordingly, while ZFNs are disclosed in the present examples, and ZFNs exhibited highly desirable properties, the present invention is not be restricted to the use of ZFNs.

[0095] ZFN technology is described extensively in the literature and, inter alia, in the following patent documents: U.S. Pat. Nos. 6,479,626, 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, 6,479,626, 8,106,255, 20030232410, and 20090203140, all of which are incorporated by reference. ZFNs can be obtained commercially from Sigma-Aldrich (St. Louis, Mo., US) under the CompoZr Zinc Finger Nuclease Technology branded products and services.

[0096] TALEN technology is described extensively in the literature and, inter alia, in the following patent documents: U.S. Pat. No. 8,420,782, U.S. Pat. No. 8,470,973, U.S. Pat. No. 8,440,431, U.S. Pat. No. 8,440,432, U.S. Pat. No. 8,450,471, U.S. Pat. No. 8,586,363, U.S. Pat. No. 8,697,853, EP2510096, U.S. Pat. No. 8,586,526, U.S. Pat. No. 8,623,618, EP2464750, US2011041195, US2011247089, US2013198878, WO2012/116274, WO2014110552, WO2014070887, WO2014022120, WO2013192316, and WO2010008562, all of which are incorporated by reference. TALENs can be obtained commercially from Thermo Fisher Scientific, Inc. (Waltham, Mass., US) under the GeneArt TALs branded products and services (formerly marketed under the Life Technologies brand).

[0097] CRISPR/Cas technology is described extensively in the literature (e.g. Cong et al. Multiplex Genome Engineering Using CRISPR/Cas Systems, Science, 15 Feb. 2013: Vol. 339 no. 6121 pp. 819-823) and, inter alia, in the following patent documents: U.S. Pat. No. 8,697,359, US2010076057, WO2013/176772, U.S. Pat. No. 8,771,945, US2010076057, US2014186843, US2014179770, US2014179006, WO2014093712, WO2014093701, WO2014093635, WO2014093694, WO2014093655, WO2014093709, WO2013/188638, WO2013/142578, WO2013/141680, WO2013/188522, U.S. Pat. No. 8,546,553, WO2014/089290, and WO2014/093479, all of which are incorporated by reference. CRISPR/Cas systems can be obtained commercially from Sigma-Aldrich (St. Louis, Mo., US) under the CRISPR/Cas Nuclease RNA-guided Genome Editing suite of products and services, or from Thermo Fisher Scientific, Inc. (Waltham, Mass., US) under the GeneArt CRISPR branded products and services.

[0098] A robust combination of efficient on-target marking and minimal toxicity to early embryogenesis can be achieved by the delivery of nuclease-encoding mRNA to the embryo.sup.12. We transferred the ORFs encoding the RELA ZFNs into two distinct vectors for in vitro mRNA production (pVAX, which requires in vitro polyadenylation, and pGEM, which contains a polyA track of defined length). For both vectors, we generated constructs bearing single ZFNs, and constructs bearing both ZFNs on the same ORF separated by a self-cleaving 2A signal. Capped and polyadenylated mRNA was then in vitro transcribed from all constructs, and the on-target editing efficiency assessed by transient transfection into pig PK15 cells.

[0099] This was followed by Surveyor/Cel-1 and a deep-sequencing based assay to measure the percentage of edited chromatids. Robust editing efficiency, in all cases exceeding that driven by positive control ZFNs, was obtained with all four vector/ORF configurations. We have previously shown that delivery of engineered nucleases to the cytoplasm of livestock zygotes can result in the production of small insertions or deletions (indels) due to non-homologous end-joining-driven (NHEJ) break repair at the target site.sup.13-15. It was not clear whether this delivery method could also result in HDR if combined with a DNA template. We co-injected porcine zygotes with mRNA encoding the pair of ZFN and either a single stranded oligodeoxynucleotide (ssODN.sup.16) or plasmid DNA bearing the warthog SNPs. Injected zygotes were transferred to recipient gilts.sup.14.

[0100] To determine whether the nucleases drove targeted editing of pig RELA, ear notches were taken from piglets 2 days postpartum and genomic DNA was prepared. PCR spanning the target locus and sequencing of these products was used to identify either alleles bearing small indels (a result of NHEJ) or specific point mutations (a result of HDR events). Highly surprisingly, no indels were observed at the ZFN target site in any of the animals genotyped; this contrasts both with our ability to obtain edited animals bearing NHEJ-generated alleles, and our earlier experience with genome editing by indels in pig.sup.13, 14.

[0101] The lack of indels at the nuclease target site was not due to a failure of the genome editing process itself, as four live piglets bore HDR-generated alleles of RELA (see Table 2 below).

[0102] All four occurred in the cohort of 46 animals injected with ZFN-encoding mRNA and a plasmid repair template. In contrast, no HDR events were observed in the 39 live born pigs where ssODN was provided as the HDR template. Sanger sequencing of PCR products spanning the target locus of the HDR positive pigs showed that piglets 354 and 364 were heterozygous at each of the 5 base changes encoded by the plasmid template, thus representing full haplotype introgression (FIG. 2). Piglet 367 was homozygous for 4 base changes proximal to the ZFN target site, and heterozygous for the most distal modification. This finding demonstrated that continuity of gene conversion tracks from DSB-R in mammalian cells, and indicates that the two homologs were cleaved in the early embryo followed by distinct HDR-based resolution of the break. Remarkably, piglet 563 was homozygous for all 5 base changes.

[0103] Livestock breeding has enabled a continuous increase in animal productivity since animal domestication. The challenge ahead is to accelerate this improvement process to meet the demands imposed on agriculture through climate change, resource and land availability in conjunction with the increase in human population. Genome editing technology has the potential to revolutionize livestock breeding.sup.4, and targeted gene knockout in several livestock species has been attained using multiple distinct designed nuclease platforms, including ZFNs, TAL effector nucleases, and CRISPR/Cas9.sup.11, 13, 15, 17-19.

[0104] The present inventors have significantly expanded the genome editors' toolbox to include the targeted transfer of an entire haplotype. Specifically, through homology dependent repair of a ZFN-induced break using a plasmid repair template we have introgressed an allele of the RELA gene between swine species, producing live piglets both heterozygous and homozygous for the desired haplotype.

Materials and Methods

ZFN Design and Validation.

[0105] ZFNs against the indicated position of the pig RELA gene were designed and assembled using an archive of pre-validated two-finger modules as described.sup.[2]. The ORFs were cloned into expression vectors harbouring enhanced obligate heterodimer forms of Fokl.sup.[20] optimized for delivery in DNA form and for production of in vitro transcribed mRNA (Vierstra et al., in press). ZFN target sequences and DNA recognition helices are described in Table 1. Pig PK15 cells were electroporated using ZFN-encoding DNA or mRNA as described, genomic DNA harvested 48 h following electroporation, and percentage of chromatids bearing indels was measured using Surveyor/Cel1 as described.sup.[10] or deep sequencing on the Illumina platform.

TABLE-US-00003 TABLE1a ZFNtargetsequences(SEQIDNOsareprovidedinbrackets) ZFNBindingSequence(underlined) TargetingZFNID AGAGGCCCTGCTGCAGCTGCAGTTTGATACTGATGAGGACC(1) 48307 TCTCCGGGACGACGTCGACGTCAAACTATGACTACTCCTGG(2) 48304

TABLE-US-00004 TABLE1b ZFNDNArecognitionhelices ZFNID Finger1 Finger2 Finger3 Finger4 Finger5 48307 DRSDLSR(3) RSDNLTR(4) TSGNLTR(5) LRQDLNK(6) TSSNLSR(7) 48304 AMQTLRV(8) DRSHLAR(9) RSDNLSE(10) KRCNLRC(11) RSAVLSE(12)

Design and Construction of HDR Templates.

[0106] A 96-mer ssODN was designed spanning the target site of the ZFN and containing two base changes encoding the desired T448A conversion (FIG. 1) plus a third (silent) base change to assist in preventing ZFN re-cutting of any introgressed alleles. The plasmid DNA template was designed with the same three base changes as the ssODN at the ZFN target site, with additional single base changes encoding the S485P and S531P (FIG. 1). This 251 bp central domain containing all five base changes was flanked by homology arms of 626 bp and 799 bp, 5 to the first base change and 3 to the final base change respectively.

Zygote Injection and Transfers.

[0107] Embryos were produced from Large-White gilts that were approximately 9 months of age and weighed at least 120 kg at time of use. Super-ovulation was achieved by feeding, between day 11 and 15 following an observed oestrus, 20 mg altrenogest (Regumate, Hoechst Roussel Vet Ltd) once daily for 4 days and 20 mg altrenogest twice on the 5th day. On the 6th day, 1500 IU of eCG (PMSG, Intervet UK Ltd) was injected at 20:00 hrs. Eighty-three hours later 750 IU hCG (Chorulon, Intervet UK Ltd) was injected. Donor gilts were inseminated twice 6 hours apart after exhibiting heat generated following super-ovulation. Embryos were surgically recovered from mated donors by mid-line laparotomy under general anaesthesia on day 1 following oestrus into NCSU-23 HEPES base medium. Embryos were subjected to a single 2-5 pl cytoplasmic injection of the pVAX single mRNAs at 2 ng/L or 4 ng/L with ssODN or plasmid template respectively. Recipient females were treated identically to donor gilts but remained un-mated. Following ZFN injection, fertilized embryos were transferred to recipient gilts following a mid-line laparotomy under general anaesthesia. During surgery, the reproductive tract was exposed and embryos were transferred into the oviduct of recipients using a 3.5 French gauge tomcat catheter. Litter sizes ranged from 1-13 piglets.

TABLE-US-00005 TABLE 2 Summary of pig zygote injections No zygotes N.sup.o live Construct injected N.sup.o recipients piglets NHEJ HDR ZFN + ssODN 95 4 39 0 0 ZFN + plasmid 272 6 46 0 4

Genotyping.

[0108] Genomic DNA was prepared from ear biopsy taken from piglets 2 days postpartum. PCR amplification with AccuPrime HiFi was conducted with primers oSL1 (gggtacaaagaggggtgaggSEQ ID NO 13) which binds out-with the 5 homology arm encoded by the plasmid and oSL2 (ctagctctgccctttccagaSEQ ID NO 14) which binds within the 3 homology arm of the plasmid. Cycling was 95 C. for 120 seconds then 40 cycles of 94 C. for 30 seconds, 59 C. for 30 seconds and 68 C. for 90 seconds, followed by primer extension of 68 C. for 5 minutes. Purified PCR products were directly sequenced.

Reference Sequences

[0109] Sus scrofa v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), mRNA/cDNA sequence (NCBI Accession NM_001114281, Version NM_001114281.1), ZFN binding site is underlined and cut site is located between the two bases shown in bold.

TABLE-US-00006 (SEQIDNO15) 1 atggacgacctcttccccctcatcttcccctcggagccggccccggcctcgggcccctat 61 gtggagatcatcgagcagcccaagcagcggggcatgcgcttccgctacaagtgcgagggc 121 cgctcagccggcagtatcccgggcgagaggagcacggataccaccaagacccaccccacc 181 atcaagatcaatggctacacggggccagggacagtgcgcatctccctggtcaccaaggac 241 ccccctcaccggcctcacccccatgagctcgtggggaaagactgccgggatggcttctat 301 gaggctgagctctgcccagaccgctgcatccacagcttccagaacctggggatccagtgt 361 gtaaagaagcgggacctggaacaggccatcaatcagcgcatccagaccaacaacaacccc 421 ttccaagttcccatagaagagcagcgcggggactacgacctgaatgctgtgcggctctgc 481 ttccaggtgacagtgcgggacccagcaggcaggcccctccgcctgccgcctgtcctctct 541 caccccatctttgacaaccgtgcccccaacactgcagagctcaagatctgccgggtgaat 601 cggaactcggggagctgccttgggggcgatgagatcttcctgctgtgcgacaaggtgcag 661 aaagaggacatcgaggtgtatttcacgggcccgggctgggaggcccgaggctccttttca 721 caagccgacgtgcaccgacaagtggccatcgtgttccggacgcctccctacgcggacccc 781 agcctgcaggcccccgtgcgcgtctccatgcagctgcggcggccttcggatcgggagctc 841 agcgagcccatggaattccagtacttgccagacacagatgaccggcaccggattgaggag 901 aaacgcaaaaggacctatgagacctttaagagcatcatgaagaagagtcctttcaatgga 961 cccaccgacccccggcctgcaacccggcgcattgctgtgccttcccgcagctcagcttcc 1021 gtccccaagccagctccccagccctatccctttacgccatctctcagcaccatcaacttt 1081 gacgagttcacgcccatggcctttgcttctgggcagatcccaggccagacctcagccttg 1141 gccccagcccctgccccagtcctggtccaggccccagccccggccccagccccagccatg 1201 gcatcagctctggcccaggccccagcccctgtccccgtcctagcccccggccttgctcag 1261 gctgtggccccgcctgcccctaaaaccaaccaggctggggaagggacactgacagaggcc 1321 ctgctgcagctgcagtttgatactgatgaggacctgggggccctgctcggcaataacact 1381 gacccgaccgtgttcacggacctggcatccgtcgacaactctgagtttcagcagctgctg 1441 aaccagggtgtatccatgcccccccacacagctgagcccatgctgatggagtaccctgag 1501 gctataactcgcttggtgacagggtcccagagaccccctgacccagctcccactcccctg 1561 ggggcctctgggctcaccaacggtctcctctcgggggacgaagacttctcctccattgcg 1621 gacatggacttctcagcccttctgagtcagatcagctcctaa SusscrofaRELA,proteinsequence(NCBIReferenceSequence:NP_001107753.1): (SEQIDNO16) MDDLFPLIFPSEPAPASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIK INGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKR DLEQAINQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPAGRPLRLPPVLSHPIFDN RAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQV AIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFK SIMKKSPFNGPTDPRPATRRIAVPSRSSASVPKPAPQPYPFTPSLSTINFDEFTPMAFASGQ IPGQTSALAPAPAPVLVQAPAPAPAPAMASALAQAPAPVPVLAPGLAQAVAPPAPKTNQAGE GTLTEALLQLQFDTDEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVSMPPHTAEPMLM EYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLSGDEDFSSIADMDFSALLSQISS

REFERENCES

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