Genetically Edited Animal

Abstract

The present invention relates to a genetically edited animal, especially to a genetically edited pig in which expression or activity of the RELA protein has been modified. Such pigs have at least partial protection against the African Swine Fever Virus. The invention also provides, a cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell of a non-human animal comprising a genetic modification which alters the expression or function of RELA protein, methods for editing the genome of animals and methods for screening the efficacy of a pharmaceutical agent in such an animal.

Claims

1. A genetically edited pig comprising a genetic modification to the sequence of the RELA gene which alters the expression or activity of the RELA protein, wherein the modification results in the following changes in the amino acids of the RELA protein: T448A, S485P and S531P.

2-3. (canceled)

4. The pig of claim 1 in which all cells of the pig contain the genetic modification.

5-10. (canceled)

11. The pig of claim 1 in which the modification is bi-allelic.

12-18. (canceled)

19. The pig of claim 1 wherein the RELA gene has been edited such that it comprises a sequence which encodes a RELA protein with a sequence as follows: TABLE-US-00010 (SEQIDNO9) LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHT AEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIA DM.

20. The pig of claim 1 wherein at least a portion of the autologous RELA sequence has been replaced with a sequence which encodes the corresponding warthog (Phacochoerus sp.) RELA protein sequence.

21. The pig of claim 1 in which a portion of the autologous RELA gene has been removed and the following corresponding sequence, or a variant thereof which encodes the same polypeptide, has been inserted: TABLE-US-00011 (SEQIDNO11) GTTTGATgCTGATGAGGACCTGGGGGCCCTGCTCGGCAATAACACTGACCC GACCGTGTTCACGGACCTGGCATCCGTCGACAACTCTGAGTTTCAGCAGCT GCTGAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGAT GGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCC CtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTcGACC CTCCTCcCcGGGGACGAgGACTTC

22. The pig of claim 1 which demonstrates one or more of the following phenotypes: an altered NFkappaB signalling capacity; an altered disease resilience or tolerance; an altered immune response; and an altered stress response.

23. The pig of claim 1 which demonstrates improved tolerance to one or more of: virus infection; pathogen infection, other than viral infection; and general or specific stressors.

24. The pig of claim 1 which has improved tolerance to ASFV infection.

25-45. (canceled)

Description

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0105] The invention will be further described by way of the following examples, which are in no way intended to limit the scope of the invention. The examples refer to the accompanying drawings, in which:

[0106] FIG. 1 shows the Sus scrofa RELA cDNA sequence, GenBank accession number NM_001114281 (SEQ ID NO 1).

[0107] FIG. 2 shows the Sus scrofa RELA amino acids sequence, GenBank accession number NP_001107753 (SEQ ID NO 2).

[0108] FIG. 3 shows the Sus scrofa RELA primary amino acid sequence showing allelic variation in transactivation domains and Rel homology domain (see also Palgrave et al, 2011). Domestic pig sequence EMBL deposited number FN999988. Warthog pig sequence EMBL deposited number FN999989. The full domestic pig (Sus scrofa) sequence is provided and underneath, positions where the warthog sequences differs are indicated, i.e. T448A, S485P and S531P (SEQ ID NO 18).

[0109] FIG. 4 shows the Sus scrofa genomic sequence showing the editor binding sites in bold. Sequence locations marked refer to porcine RELA cDNA, Accession number NM_001114281; fully genomic Sus scrofa sequence accession number NC_010444, specifically between 5699452 and 5707267 of Assembly 10.2 (SEQ ID NOS 19 and 29).

[0110] FIG. 5 shows a diagram of the Sus scrofa RELA genomic sequence (SEQ ID NO 37) with TALEN (boxes; TALEN11-1L and TALEN11-1R) and ZFN (boxes ZFN1 and ZFN2) binding sites identified; relative positioning of PCR primers also shown (boxes ss p65NJF1 and ssp65NJR1).

[0111] FIG. 6 shows gel electrophoresis identification of editing events by Cell assay. Amplified products were digested with Cell and assessed by gel electrophoresis. Minor bands lower down the gel below dominant band indicate mismatch and editing event. The first 2 gels identify heterozygous (one allele) editing events; third gel identifies homozygous (bi-allelic events) through sample mixing assay.

[0112] FIG. 7 shows exemplar sequence of RELA edited site (indel). Sequencing trace and sequence interpretation of indels at porcine RELA in two individual pigs (#16 and #26) are shown (SEQ ID NOS 32 to 36, relative to order shown in Fig).

[0113] FIG. 8 shows the location of TALEN, ZFN and PCR primers for sequencing amplicon; porcine RELA sequence 769417-769110 accession number NW_003609513.1 (SEQ ID NO 37).

[0114] FIG. 9 shows an example Cell Assay; lanes A2, A12, B6, B14 and B20 were identified as having cleavage products consistent with editing events at the target site. Direct sequencing of the same PCR products confirmed this interpretation and identified B12, B16, B17 and B18 as having only wild-type sequence.

[0115] FIG. 10 shows specific indels identified in several piglets (SEQ ID NOS 19 to 31).

EXAMPLE 1: EDITOR DESIGN AND CONSTRUCTION

[0116] Two types of editor were used: TALEN and ZFN. Both were designed to target the same region of porcine RELA gene.

[0117] TALEN: All TALENs were designed using the TALE-NT software and assembled using methods described in Cermak et al. (2011)Nucl. Acids Res. (2011) 39 (12): e82. Briefly, intermediary arrays were produced for each TALEN pair that were compatible for Golden Gate cloning into pC-+63-TAL modified vector (although other vectors such as pC-+231-TAL, RCIscript-+231-TAL, pC-GoldyTALEN or RCIscript-GoldyTALENetc. could be used). Arrays were joined in the above vectors as follows; 150 ng each pFUS_A, pFUS_B, pLR-X and the desired backbone were mixed in a 20 l digestion/ligation reaction including 50 units T4 DNA ligase (New England Biolabs) and 10 units Esp3I (Fermentas) in 1T4 ligase buffer (New England Biolabs). The reaction was incubated in a thermocycler for 10 cycles of 5 min at 37 C. and 10 min at 16 C., then heated to 50 C. for 5 min and then 80 C. for 5 min. Two microliters of each reaction was transformed into E. coli and plated on LB-carbenicillin plates. Plasmid DNA was purified and mRNA synthesized from SacI linearized RCIscript vectors using the mMessage Machine T3 Kit (Ambion).

[0118] The target sequences for the TALENs used in this work are shown in FIGS. 4 and 5. It is of course possible that TALENs targeting other sequences within the RELA gene, or indeed elsewhere in the pig genome, could be used in the present invention. The person skilled in the art could readily construct TALENs adapted to target essentially any other desired sequence using the same techniques described above.

[0119] ZFN: ZFNs targeted to porcine RELA were purchased from Sigma who use Sangamo algorithm to assist design of modules from the Sangamo archive to assemble the zinc finger proteins and attach the FokI nuclease domain to create the ZFN. The ZFN was supplied by Sigma as mRNA.

[0120] ZFNs targeted at other sequences of interest can also be commercially sourced.

[0121] A useful summary of gene editing using site specific nucleases can be found in the recent review by Moira A McMahon, Meghdad Randar & Matthew Porteus, Gene editing: not just for translation anymore, Nature Methods, Vol. 9, No. 1, January 2012.

EXAMPLE 2: TARGET SITE DETAILS

[0122] Target sites for the designed TALENs and ZFNs (FIGS. 4 and 5) are shown with reference to the nucleic acid numbering system of the RELA cDNA (as shown in FIG. 1), GenBank accession number NM_001114281 (SEQ ID NO.: 1); the TALEN target site is located between bases 1458 to 1505; the ZFN target site is located between bases 1496 to 1432. The full genomic Sus scofa genomic sequence NCBI Reference Sequence accession number is NC_010444 Assembly 10.2.

EXAMPLE 3: PRODUCTION OF GENE EDITED EMBRYOS

[0123] To establish the frequency of gene editing in pig embryos an in vitro embryo culture experiment was performed.

[0124] Pig ovaries were collected, washed with pre-warmed phosphate buffered saline at 38.5 C. and follicles aspirated. Oocytes were washed in TL HEPES PVA before culturing in maturation medium for 44 hours (22 hours plus hormones and 22 hours minus hormones; 38.5 C., 5% CO.sub.2), followed by gentle pipetting to remove cumulus cells and incubation with prepared sperm for 6 hours (38.5 C. in 5% CO.sub.2). Zygotes were transferred to NCSU-23 HEPES base medium and subjected to a single 2-10 l cytoplasmic injection of mRNA at 2, 10 or 25 ng/l. The 25 ng/l mRNA sample consisted of 20 ng/l of either a TALEN or ZFN pair mRNA and 5 ng/l EGFP mRNA. The 10 and 2 ng/l mRNA samples were dilutions of the 25 ng/l sample. Zygotes were cultured in batches for 66 hours (embryo culture medium; 5% CO2, 38.5 C.), following which they were placed individually into micro-droplets of medium under oil for visual inspection or harvested for genotyping.

[0125] The results of these experiments are shown in Table 1 below.

TABLE-US-00006 TABLE 1 Frequency of editing events TALEN injections Embryos No edited % edited/analysed % edited/born No. embryos analysed 120 25 21% No. pigs analysed 46 8 17% ZFN injections No. pigs analysed 9 1 11%

EXAMPLE 4: GENOTYPING OF EDITING EVENTS IN EMBRYOS

[0126] Gene editing events in porcine embryos were identified by direct sequencing of amplified, isolated DNA and through a gel electrophoresis assay. The latter identified mismatch between the two alleles through digestion by the Cell enzyme.

[0127] DNA was amplified from harvested embryos using the REPLI-g mini kit, Qiagen. The REPLI-g DNA sample was then used as a template for High fidelity PCR (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) using single-stranded oligonucleotidep65NJF1 (GCAATAACACTGACCCGACCGTG) (SEQ ID NO 16) and single-stranded oligonucleotidep65NJR1 (GCAGGTGTCAGCCCTTTAGGAGCT) (SEQ ID NO 17) as primers designed to amplify a 308 base pair region (see FIG. 5) of the wild-type porcine RELA gene that overlapped the TALEN and ZFN cut sites. The PCR product was then sent for sequence analysis to allow identification of editing events. Alternatively, the PCR products were cloned into a plasmid and individual plasmids sequenced allowing heterozygous and mosaic editing events to be analysed separately. From 56 of the EGFP positive embryos, 16 were confirmed to harbour RELA editing events (often termed an indel). Of these edited embryos, 10 harboured a heterozygous mutation in one allele, while the remaining 6 carried a mutation on both alleles.

[0128] The presence of mutations in the RELA gene were additionally identified using a Cell assay (SURVEYOR mutation detection kit, TRANSGENOMIC). The high fidelity PCR product was denatured/re-annealed before being subjected to SURVEYOR nuclease activity which cuts at base mismatches highlighting insertions, deletions and substitutions. The resulting fragments were subsequently separated by gel electrophoresis for analysis with size differences identifying edited indel events.

EXAMPLE 5: GENERATION OF GENE EDITED PIGS

[0129] Gene edited pigs were produced through injection of the TALEN or ZFN mRNA into the cytoplasm of porcine zygotes.

[0130] 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.

[0131] 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 l cytoplasmic injection of either ZFN or TALEN pair mRNA at 2 ng/l. Recipient females were treated identically to donor gilts but remained un-mated. Following TALEN or 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.

EXAMPLE 6: GENOTYPING OF EDITING EVENTS IN PIGS

[0132] Gene editing events in born piglets were identified by direct sequencing of amplified, isolated DNA and through gel electrophoresis assay. The latter identified mismatch between the two alleles through digestion by the Cell enzyme.

[0133] The DNA was extracted from tissue samples (e.g. ear skin biopsy) using the DNeasy Blood and Tissue kit, QIAGEN. A sample of purified DNA was then used as a template for High fidelity PCR (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) using single-stranded oligonucleotide p65NJF1 (GCAATAACACTGACCCGACCGTGSEQ ID NO 16) and single-stranded oligonucleotide p65NJR1 (GCAGGTGTCAGCCCTTTAGGAGCTSEQ ID NO 17) as primers designed to amplify a 308 base pair region (FIG. 5) of the wild-type porcine RELA gene that overlapped the TALEN and ZFN cut sites. The PCR product was then sent for sequence analysis to allow identification of editing events. Alternatively, the PCR products were cloned into a plasmid and individual clones sequenced allowing heterozygous and mosaic editing events to be analysed separately.

[0134] The presence of mutations in the RELA gene were additionally identified using a Cell assay (SURVEYOR mutation detection kit, TRANSGENOMIC). The high fidelity PCR product was denatured/re-annealed before being subjected to SURVEYOR nuclease activity which cuts at base mismatches highlighting insertions, deletions and substitutions. The resulting fragments were subsequently separated by gel electrophoresis for analysis with size differences identifying edited indel events.

[0135] From 46 born piglets analysed following TALEN pair zygotic injection, 8 were identified as being genome edited (Table 1). See FIG. 7 for exemplars of direct sequence analysis from 2 of the TALEN genome edited piglets. From 9 born piglets analysed following ZFN pair zygotic injection 1 was identified as being genome edited (Table 1).

[0136] The following examples relate to methodology to create pigs with sequences inserted into the RELA gene and to analyse the phenotype of genetically edited pigs.

EXAMPLE 7: PRODUCTION OF GENE INTROGRESSION EMBRYOS USING EDITING TECHNOLOGY

[0137] To establish the frequency of gene editing in pig embryos in vitro embryo culture experiment is performed.

[0138] Pig ovaries are collected, washed with pre-warmed phosphate buffered saline at 38 C. and follicles aspirated. Oocytes are washed in TL HEPES PVA before culturing in maturation medium for 44 hours (22 hours plus hormones and 22 hours minus hormones; 39 C., 5% CO.sub.2), followed by gentle pipetting to remove cumulus cells and IVF for 6 hours (38.5 C. in 5% CO.sub.2). Zygotes are transferred to NCSU-23 HEPES base medium and subjected to a single 2-5 l cytoplasmic or pronuclear injection of either ZFN or TALEN pair mRNA between 2 to 10 ng/l mixed with from 1 to 10 ng/l (optimum concentrations can be determined by experimenter) single-stranded or double-stranded DNA fragment or plasmid. Zygotes are cultured in batches for 66 hours (embryo culture medium; 5% CO.sub.2, 38.5 C.), following which they are placed individually into micro-droplets of medium under oil for visual inspection or harvested for genotyping.

EXAMPLE 8: GENOTYPING OF GENE INTROGRESSION EVENTS IN EMBRYOS

[0139] Gene introgression events in porcine embryos are identified by direct sequencing of amplified, isolated DNA.

[0140] DNA is amplified from harvested embryos using the REPLI-g mini kit, Qiagen. The REPLI-g DNA sample is then used as a template for High fidelity PCR (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) using single-stranded primers designed to amplify replicon containing the target region. The PCR product is then sent for sequence analysis to allow identification of editing events. Alternatively, the PCR products are cloned into a plasmid and individual plasmids sequenced allowing heterozygous and mosaic editing events to be analysed separately.

EXAMPLE 9: PRODUCTION OF GENE INTROGRESSION PIGS USING EDITING TECHNOLOGY

[0141] Gene introgression edited pigs are produced through injection of the TALEN or ZFN mRNA in combination with a single-stranded DNA oligo or double-stranded DNA fragment into the cytoplasm or nucleus of porcine zygotes.

[0142] Embryos are produced from Large-White gilts that are approximately 9 months of age and which weigh at least 120 kg at time of use. Super-ovulation is 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) is injected at 20:00 hrs. Eighty three hours later 750 IU hCG (Chorulon, Intervet UK Ltd) is injected.

[0143] Donor gilts are inseminated twice 6 hours apart after exhibiting heat generated following super-ovulation. Embryos are surgically recovered from mated donors by mid-line laparotomy under general anaesthesia on day 1 following oestrus into NCSU-23 HEPES base medium. Embryos are subjected to a single 2-5 l cytoplasmic or pronuclear injection of either ZFN or TALEN pair mRNA at 2 to 10 ng/l mixed from 1 to 10 ng/l (optimum concentrations can be determined by experimenter) single-stranded or double-stranded DNA fragment or plasmid.

[0144] Suitable sequences are:

TABLE-US-00007 (SEQIDNO10) GAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGA aTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCcGA CCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCTCcC cGGGGACGAgGACTTC (SEQIDNO11) GTTTGATgCTGATGAGGACCTGGGGGCCCTGCTCGGCAATAACACTGACCC GACCGTGTTCACGGACCTGGCATCCGTCGACAACTCTGAGTTTCAGCAGCT GCTGAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGAT GGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCC cGACCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCT CcCcGGGGACGAgGACTTC

[0145] Recipient females are treated identically to donor gilts but remain un-mated. Following TALEN or ZFN plus DNA injection, fertilized embryos are transferred to recipient gilts following a mid-line laparotomy under general anaesthesia. During surgery, the reproductive tract is exposed and embryos are transferred into the oviduct of recipients using a 3.5 French gauge tomcat catheter.

EXAMPLE 10: GENOTYPING FOR GENE INTROGRESSION IN EDITED PIGS

[0146] Gene introgression events in born piglets are identified by direct sequencing of amplified, isolated DNA.

[0147] The DNA is extracted from tissue samples (e.g. ear skin biopsy) using the DNeasy Blood and Tissue kit, QIAGEN. A sample of purified DNA is then used as a template for High fidelity PCR (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) using single-stranded oligonucleotides p65NJF1 (GCAATAACACTGACCCGACCGTGSEQ ID NO 16) and single-stranded oligonucleotide p65NJR1 (GCAGGTGTCAGCCCTTTAGGAGCTSEQ ID NO 17) designed to amplify a 308 base pair region (FIG. 5) of the wild-type porcine RELA gene that overlapped the TALEN and ZFN cut sites. The PCR product was then sent for sequence analysis to allow identification of editing events. Alternatively, the PCR products were cloned into a plasmid and individual clones sequenced allowing heterozygous and mosaic editing events to be analysed separately.

EXAMPLE 11: EVALUATION OF ALTERED RELA LEVELS IN PIG TISSUE AND IN CELLS ISOLATED FROM GENE EDITED PIGS

[0148] To assess the effect of editing of the RELA locus on RELA levels one can utilise qRT-PCR and RELA protein levels by Western blot and ELISA in pig tissue (e.g. skin) or cells (e.g. primary fibroblasts isolated from skin biopsy, PBMCs isolated from blood) isolated from edited pigs. Suitable primers and probes for qRT-PCR can readily be determined by the person skilled in the art.

EXAMPLE 12: EVALUATION OF ALTERED NFKAPPAB SIGNALLING IN PIG TISSUE AND IN CELLS ISOLATED FROM GENE EDITED PIGS

[0149] RELA is a predominant component of the NFkappaB transcription factor. To assess the effect of editing of the RELA locus on NFkappaB signalling one can perform qRT-PCR for genes known to be activated by NFkappaB on pig tissue (e.g. skin) or cells (e.g. primary fibroblasts isolated from skin biopsy, PBMCs isolated from blood) isolated from edited pigs with or without stimulation, e.g. by LPS, TNFalpha, CSF1.

EXAMPLE 13: EVALUATION OF ALTERED CELLULAR RESPONSE TO VIRUS CHALLENGE IN CELLS ISOLATED FROM GENE EDITED PIGS

[0150] To assess the effect of editing of the RELA locus on the cellular response to virus challenge PBMCs from blood are isolated. Cultured PBMCs, either with or without CSF1, are exposed to virus challenge (e.g. influenza virus, PRRSV). The signalling response to virus challenge is assessed by expression profiling.

CONCLUSION

[0151] There have been described a number of methodologies to modify (edit) the genome of pigs. These methodologies can readily be adapted to modify the genetics of other animals, such as cows, sheep, goats and chickens.

[0152] In pigs the modification of RELA can provide increased tolerance against ASFV. This provides a novel mechanism by which tolerance to this extremely significant pathogen can be created. The commercial and ecological importance of this is great. Domestic pig production in Africa is severely restricted because of the effects of ASFV. Outside of Africa, ASFV is endemic in feral pigs in Sardinia, Italy. In 2007 it was introduced into the Caucasus, and has apparently become endemic among wild boars in the region. There have been outbreaks of the virus in domesticated swine in the Republic of Georgia, Russia, Armenia, Azerbaijan and other countries in the region. There is thus a great need to protect against the spread of this disease and also to mitigate the risk should controlling prove impossible. The present invention has the potential to significantly mitigate the problems should ASFV infection become more widespread and raises the possibility of increasing pig production in Africa.

ADDITIONAL EXEMPLIFICATION

[0153] Additional work was conducted to further demonstrate the power of the above mentioned gene editing techniques. In this work it is demonstrated that both TALEN and ZFN technology can be efficiently applied to engineer pig zygotes that result in gene edited live births, both mono- and bi-allelic (Table 2), significantly broadening the use of editor technology in livestock.

[0154] We performed cytoplasmic editor injection into porcine zygotes, given the difficulty associated with visualization of the pronucleus in porcine zygotes. Oocytes were harvested from slaughterhouse material for in vitro studies and superovulated artificially inseminated sows for embryos destined for transfer into recipients. Initially 208 zygotes were subjected to a single cytoplasmic injection of 10 l of a solution composed 20 ng/l RELA TALEN mRNA with 5 ng/l EGFP mRNA (to enable visual identification and isolation of embryos that functionally translated the injected mRNA). After approximately 3 days of in vitro development, GFP fluorescence was detected in 36% of embryos. Thus mRNA injected into the cytoplasm of pig zygotes translates to functional protein in the embryo. GFP positive embryos were screened for editing events by Cell surveyor assay (FIG. 9) and sequencing of PCR amplified fragments (shown below and in FIG. 10). We detected 16 editing events in 46 GFP-positive embryos analysed (35%). In a second experiment we tested 34 embryos injected with 2 l of 20 ng/l RELA TALEN mRNA but without selection for GFP activity, and detected 2 editing events (6%). In two further experiments where 2 l of 10 ng/l or 2 g/l RELA TALEN mRNA was injected, 0% and 18% editing frequency, respectively, was observed. Thus, in total we identified 21 editing events in porcine embryos in vitro (21% of tested embryos), and a high frequency of these editing events were biallelic in nature (29% of editing events). Calculating this as a frequency of tested embryos, we achieved a biallelic editing frequency of 6%.

[0155] Since both the highest and lowest tested concentrations of RELA TALEN mRNA produced edited embryos in vitro we elected to transfer zygotes injected with each tested TALEN amount into recipient sows and allow pregnancies to develop to term. Pregnancy rates for the higher concentrations of RELA TALEN mRNA were poor; 1 out of 2 transfers and 0 out of 2 transfer for embryos injected with 10 ng/l or 20 ng/l, respectively. This poor pregnancy rate reflect the visual observation that 2 ng/l RELA TALEN mRNA injected embryos developed better in vitro than those injected with higher concentrations of TALEN mRNA. The one pregnancy from 10 ng/l delivered 7 piglets, none of which harboured RELA editing events by direct sequencing of PCR amplified products. We did not pursue transfers of embryos with these higher TALEN mRNA concentrations any further.

[0156] In contrast, transfer of embryos injected with 2 ng/l RELA TALEN RNA resulted in 5 pregnancies from 7 recipients. One subsequently aborted at 15 weeks of pregnancy just prior to parturition; analysis of the 9 foetuses carried revealed 3 to have editing events. In total from the remaining 4 farrowings, 39 piglets were produced of which 8 carried editing events (21%). Of the 8 editing events, 2 animals were stillborn and a further 1 died neonatally due to being crushed by the mother; 5 are still alive.

[0157] In parallel we tested a ZFN with a target location of 1496 to 1532 bp relative to the translational start site in porcine RELA cDNA sequence (NM_001114281) (SEQ ID NO.: 1). Again the one transfer of embryos injected with RELA ZFN mRNA at 10 ng/l failed to generate a pregnancy while the two transfers of embryos injected with RELA ZFN mRNA at 2 ng/l both became pregnant resulting in the birth of 9 piglets. Of these 9 piglets, one carried an editing event at the ZFN target site (11%); although low numbers, this is a comparable frequency in comparison to our observed TALEN editing efficiency.

[0158] Direct sequencing of PCR products revealed a variety of editing events in piglets derived from TALEN and ZFN injected embryos. Analysis of ear biopsy isolated genomic DNA identified both deletions and insertions at the target sites. Sequence data from 2 animals constituted multiple overlapping traces indicating two or more editing events; this was subsequently confirmed by sequencing of multiple cloned PCR products. Presuming that in these cases of multiple editing the frequency of events detected in ear biopsy reflects frequency in the early embryo, designer nuclease editing can remain active beyond the 2-cell stage (i.e. some events display low representation in the PCR pool and are therefore only present in a subset of cells). In total, 5 biallelic events where identified from 9 edited piglets (56%; 9% of piglets born); 4 from TALEN and 1 from ZFN mRNA injections. Of these bialleleic events 2 were homozygous with 3 displaying different indels on each allele. While both piglets carrying homozygous biallelic event survived farrowing (milk in stomach post mortem), they both died within the first 24 hours of life: the ZFN-derived piglet with an 18 bp biallelic homozygous deletion was bitten by its mother while the TALEN-derived piglet with a 6 bp biallelic homozygous deletion was crushed by its mother.

[0159] In summary, we observed an overall editing frequency of 2% of transferred embryos or 16% of piglets born. These figures compare favourably with that reported for zygote injection of ZFNs in rats where 2% of transferred embryos and 12% of founder animals harboured editing events (Guerts, A. M. et al. Science 325, 433 (2009)). Our editing frequencies also compare favourably with the production of monoallelic (0.1% of transferred embryos) and biallelic (1% of transferred embryos; the greater frequency over monoallelic due to incorporation of a FACS enrichment stage prior to somatic cell nuclear transfer (Hauschild, J. et al. Proc. Natl. Acad. Sci. USA 108, 12013-12017 (2011)) pigs using somatic cell nuclear transfer methodology.

[0160] In conclusion, we demonstrate that editor technology, both TALEN and ZFN, can be successfully applied to pig zygotes to produce live gene edited pigs and contrary to predictions the delivery of editors by the direct injection into the zygote is both efficient and able to generate biallelic mutations. This novel achievement paves the way for precise genome engineering of livestock independent of somatic cell nuclear transfer (cloning) technology.

[0161] Materials and Methods for Additional Exemplification

[0162] Editor Design and Construction

[0163] Two types of editor were used: TALEN and ZFN. Both were designed to target the same region of porcine RELA gene.

[0164] TALEN: TALENs were designed using the TALE-NT software and assembled using methods described previously (Carlson, D. F. et al. Proc. Natl. Acad. Sci. USA 109, 17382-17387 (2012)). Briefly, intermediary arrays were produced for the porcine RELA TALEN pair for Golden Gate cloning as follows; 150 ng each pFUS_A, pFUS_B, pLR-X and pC-+63-TAL modified vector were incubated for 10 cycles of 5 min at 37 C. and 10 min at 16 C., then heated to 50 C. for 5 min and then 80 C. for 5 min in the presence of 50 units T4 DNA ligase (New England Biolabs), 10 units Esp3l (Fermentas), lx T4 ligase buffer (New England Biolabs). Plasmid DNA was purified from ligated vector transformed E. coli and mRNA synthesized from SacI linearized RCIscript vectors using the mMessage Machine T3 Kit (Ambion).

[0165] ZFN: ZFNs targeted to porcine RELA were purchased from Sigma. The ZFN displayed 84.7% cutting in MEL1 assay (Sigma data sheet) and was supplied as mRNA.

[0166] Zygote Injections

[0167] To establish the frequency of gene editing in pig embryos an in vitro embryo culture experiment was performed. Pig ovaries were collected, washed with pre-warmed phosphate buffered saline at 38 C. and follicles aspirated. Oocytes were washed in TL HEPES PVA before culturing in maturation medium for 44 hours (22 hours plus hormones and 22 hours minus hormones; 39 C., 5% CO.sub.2), followed by gentle pipetting to remove cumulus cells and IVF for 6 hours (38.5 C. in 5% CO.sub.2). Zygotes were transferred to NCSU-23 HEPES base medium and subjected to a single 2-10 l cytoplasmic injection of mRNA at 2, 10 or 20 ng/l+1-5 ng/l EGFP mRNA. The 10 and 2 ng/l mRNA samples were dilutions of the 20 ng/l sample. Zygotes were cultured in batches for 66 hours (embryo culture medium; 5% CO2, 38.5 C.), following which they were placed individually into micro-droplets of medium under oil for visual inspection or harvested for genotyping either as a total group or after manual selection of GFP fluorescing embryos.

[0168] Embryo Transfers

[0169] 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.

[0170] 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 2p1 cytoplasmic injection of either ZFN or TALEN pair mRNA at 10 ng/l or 2 ng/l.

[0171] Recipient females were treated identically to donor gilts but remained un-mated. Following TALEN or 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 3-17 piglets.

[0172] Genotyping

[0173] Gene editing events in porcine embryos were identified by direct sequencing of amplified, isolated DNA and through a gel electrophoresis assay. The latter identified mismatch between the two alleles through digestion by the Cell enzyme.

[0174] Sequencing: DNA was amplified from harvested embryos using the REPLI-g mini kit, Qiagen. The REPLI-g DNA sample was then used as a template for High fidelity PCR (AccuPrime Taq DNA Polymerase High Fidelity, Invitrogen) using p65NJF1 5-GCAATAACACTGACCCGACCGTG-3 (SEQ ID NO 16) and p65NJR1 5-GCAGGTGTCAGCCCTTTAGGAGCT-3 (SEQ ID NO 17) as primers designed to amplify a 308 base pair region of the wild-type porcine RELA gene that overlapped the TALEN and ZFN cut sites. The PCR product was purified then sent for sequence analysis to allow identification of editing events. Alternatively, the PCR products were cloned into a plasmid and individual plasmids sequenced allowing heterozygous and mosaic editing events to be analysed separately.

[0175] The following sequences were determined (TALEN/ZFN target sites are shown in bold, insertions are double underlined, and deletions are shown with symbol 1:

TABLE-US-00008 TALENbindingsites (SEQIDNO19) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTCWT Piglet8770-I (SEQIDNO19) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTCWT (SEQIDNO20) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCT GAGGCTATAACTC1 Piglet8770-J (SEQIDNO19) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTCWT (SEQIDNO21) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATTGCTGATGGAGTACCC TGAGGCTATAACT+1 Piglet8770-26 (SEQIDNO19) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTCWT (SEQIDNO20) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCT GAGGCTATAACTC1 Piglet8130-saton (SEQIDNO22) GGTGTATCCATGCCCCCCCACACAGCTGA~~~~~~GCTGATGGAGTACCCT GAGGCTATAACTC6 Piglet8130-16 (SEQIDNO19) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTCWT (SEQIDNO23) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCTCCATCAGCTGATGGAGT ACCCTGAGGCTAT+5 (SEQIDNO24) GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~~~~~~~~~~~~CCCT GAGGCTATAACTC17 Insertedsequence(doubleunderlined)isduplica- tion/inversionofunderlinedsequence Piglet8784-30 (SEQIDNO25) GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~ 111 (SEQIDNO26) GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCT GAGGCTATAACTC4 Piglet8784-33 (SEQIDNO27) GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~CTGATGGAGTACCCT GAGGCTATAACTC5 (SEQIDNO22) GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~CTGATGGAGTACCCT GAGGCTATAACTC6 (SEQIDNO28) GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCT GAGGCTATAACTC4 Piglet8784-34 (SEQIDNO20) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCT GAGGCTATAACTC1 (SEQIDNO29) GGTGTATCCATGCCCCCCCACACAGCTGAGCCC+TGAGTACCCTGAGGAGTACCCTGA GGCTATA8+ 12 Doubleunderlinedportionofinsertedsequenceis inversionofunderlinedsequenceinWT. ZENbindingsite (SEQIDNO30) TGCTGATGGAGTACCCTGAGGCTATAACTCGCTTGGTGACAGGGTCCCAGA GACCCCCTGACCWT Piglet8142-C (SEQIDNO31) TGCTGATGGAGTACCCTGAGGCTATAACTC~~~~~~~~~TCTGGGA~~~~~ ~~~~CCCTGACC25+ 7 Insertedsequence(doubleunderlined)isinversion ofunderlinedsequenceinWT.

[0176] Cel1 assay: The presence of mutations in the RELA gene were additionally identified using a Cell assay (SURVEYOR mutation detection kit, TRANSGENOMIC). The high fidelity PCR product was denatured/re-annealed before being subjected to SURVEYOR nuclease activity which cuts at base mismatches highlighting insertions, deletions and substitutions. The resulting fragments were subsequently separated by gel electrophoresis for analysis with size differences identifying edited events.

[0177] Comparison of In Vitro Embryo and Piglet Editing Frequency

[0178] For RELA TALEN mRNA injected zygotes we transferred 393 embryos into 11 recipient sows, resulting in 6 pregnancies and 5 farrowings with litter sizes ranging from 3 to 17. One female aborted within last two weeks of pregnancy. Of the 46 piglets born, 5 were stillborn while of the live born 13 were savaged, sat on by the sow or culled on veterinary advice. Post mortem investigation failed to detect any common pathology associated with the dead piglets.

[0179] In comparison to previous studies describing the combination of editor technology and somatic cell nuclear transfer, zygote injection represents an efficient technology. In our 32 donor/recipient animals were used to produce 9 edited piglets. In contrast the generation of genome edited pigs by somatic cell nuclear transfer used, for example, approximately 75 donor/recipients to produce 2 edited animals (Yang, D. et al. Cell Res. 21, 979-982 (2011)). while approximately 84 donor/recipient animals to produce 11 edited piglets in a report of biallelic genome editing (Hauschild, J. et al. Proc. Natl. Acad. Sci. USA 108, 12013-12017 (2011)); assuming 20 oocytes per donor animal.

[0180] Table 2 summarises the TALEN-mediated editing events in embryos and piglets.

TABLE-US-00009 TABLE 2 Numbers for TALEN edited indels in porcine embryos in vitro and piglets. Embryos in vitro GFP PCR Injected fluorescence amplified Edited* Biallelic TALEN zygotes (visual) (tested) (% of tested) (% of tested) 20 ng/l 208 75 46 16 (35%) 5 (11%) 20 ng/l 68 ND 34 2 (6%) 1 (3%) 10 ng/l 38 ND 3 0 (0%) 0 (0%) 2 ng/l 53 ND 17 3 (18%) 1 (6%) total 367 NA 100 21 (21%) 6 (6%) Piglets Transferred Edited* Biallelic Editor embryos Recipients Pregnancies Piglets born (% of born) (% of born) 20 ng/l 60 2 0 NA NA NA TALEN 10 ng/l 67 2 1 7 0 (0%) 0 (0%) TALEN 2 ng/l 266 7 5 39 8 (21%) 4 (10%)** TALEN 10 ng/l 29 1 0 NA NA NA ZFN 2 ng/l 80 2 2 9 1 (11%) 1 (11%)*** ZFN Total 502 14 8 55 9 (16%) 5 (9%) *Edited confirmed by sequencing PCR product **Of the 4 biallelic TALEN mediated editing events, only 1 was as homozygous event. ***The 1 biallelic ZFN mediated event was homozygous. NDnot determined NAnot appropriate