WO2021144574A1

WO2021144574A1 - Gene editing of unfertilized porcine and bovine oocytes

Info

Publication number: WO2021144574A1
Application number: PCT/GB2021/050081
Authority: WO
Inventors: Namdori MTANGO; Benjamin BEATON
Original assignee: Pig Improvement Company Uk Limited
Priority date: 2020-01-14
Filing date: 2021-01-14
Publication date: 2021-07-22

Abstract

Methods for creating gene edited swine and cattle at industrial quantities are provided. The methods result in reduced rates of mosaicism as compared to traditional gene-editing methods. Animals are synchronized and unfertilized, metaphase II (MII) oocytes, are harvested from the animals (e.g., via flushing or OPU). Gene editing components are introduced into the MII oocytes. For example, the oocytes can be injected with a Cas protein and at least one guide RNA (gRNA) directed to a target gene. Injected oocytes can be incubated prior to fertilization up to the point of viability, cultured to appropriate stages, and then implanted into surrogate females for gestation. Optionally, the superovulated females can be inseminated prior to harvesting the unfertilized oocytes, and zygotes can be gene edited via conventional means while unfertilized oocytes can be gene edited via a method of the present teachings.

Description

Gene editing of unfertilized porcine and bovine oocytes CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to US Provisional Application 62/961,033, filed on January 14, 2020. 62/961,033 is hereby incorporated by reference in its entirety.

FIELD

[0002] The present invention relates to methods for creating gene-edited swine and cattle. The invention further relates to a method where gene editing reagents are introduced into an unfertilized oocyte. Embryos and animals generated using the method are also provided.

BACKGROUND

[0003] The clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas system of adaptable bacterial immunity can be used to edit animal genomes (Mali et al, Science, 2013, 339, 823-826 and Hsu et al, Cell, 2014, 157, 1262-1278). Cas proteins such as Cas9 (CRISPR-associated protein 9) are DNA endonucleases that have site-specificity determined by a guide RNA (gRNA) complex comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). The crRNA contains a sequence that is complementary to a specific site within a target gene, and the tracrRNA interacts with the Cas protein, which cuts the target gene at the specific site. Natural crRNA comprises CRISPR sequences and a spacer sequence that binds to pathogen DNA, leading to double stranded breaks in the pathogen DNA. Altering the pathogen-targeting portion of the crRNA to a sequence complementary to a target gene allows the creation of targeted edits in the target gene. The crRNA and the tracrRNA can be present in separate RNA molecules that bind to one another through base-pairing of complementary sequences. Alternatively, a single guide RNA (sgRNA) that contains both the crRNA and tracrRNA in a single RNA molecule can be used (Doudna, J.A. and Charpentier, E., Science, 2014, 346, 1258096).

[0004] One way of using CRISPR/Cas to edit the genome of an animal is by injecting a zygote with the CRISPR/Cas machinery at about five hours post-fertilization. A significant drawback of this approach, however, is that a substantial number of the resulting animals are mosaic, i.e., within a single animal, there are two or more populations of cells with different genotypes (e.g., some cells contain edits in one allele of the chromosome pair, some cells carry edits in both alleles, some cells may carry unintended edits, and some cells can carry no edits, or any combination thereof). Mosaicism is particularly undesirable when producing gene-edited animals, because the cells not carrying the desired edit (or carrying only a single copy of the desired edit) may nullify the expression of the changed trait. There is therefore a need for improved methods for editing target genes in animals, and in porcine and bovine animals in particular.

[0005] Recently, several groups have attempted to reduce mosaicism by injecting CRISPR/Cas9 into oocytes instead of zygotes (Vilarino, M., et al., Scientific Reports, 2017,7, 17472; Ma, H. et al., Nature, 2017, 538, 413-419; U.S. Patent No. 9,783,780; PCT Publication No. WO 2016/097751). However, these references indicate that reduction of mosaicism is species-dependent. None of these references demonstrate the use of this technique in pigs or cattle.

SUMMARY

[0006] Methods for production of non-mosaic gene-edited swine are provided. In various embodiments, the present disclosure provides for and includes a method of creating gene- edited swine comprising: providing at least one unfertilized oocyte from a sow or a gilt; and introducing into the at least one unfertilized oocyte: (a) a Cas protein or a nucleic acid encoding a Cas protein; and (b) at least one guide RNA (gRNA) directed to a target gene. In some configurations, the method can further comprise maturing the at least one unfertilized oocyte to metaphase II (Mil) prior to introduction of the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA.

[0007] In various configurations, the method can further comprise fertilizing the at least one unfertilized oocyte after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA.

[0008] In various configurations, the at least one oocyte can be a Sus scrofa oocyte. In various configurations, after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA, the at least one oocyte can be incubated for up to 5 hours prior to fertilization. In various configurations, after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA, the at least one oocyte can be incubated for 30 minutes to 2 hours prior to fertilization.

[0009] In various configurations, introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte can comprise injecting the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte.

[0010] In various configurations, introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte can comprise electroporating the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte. [0011] In various configurations, the at least one gRNA can comprise a single guide RNA (sgRNA). In various configurations, the at least one gRNA can comprise at least two sgRNAs.

[0012] In various configurations, the target gene can affect a health trait, a reproductive trait, or a growth trait. In various configurations, the target gene can encode a protein involved in infection of swine by a pathogen. In some configurations, the target gene can encode a protein essential for infection of swine by the pathogen.

[0013] In various configurations, the target gene can be ANP32, ANPEP, TMPRSS1, TMPRSS2, NANOS2, CD 163, Melanocortin-4 receptor (MC4R), HMGA, IGF2, E.coli F4ab/ac, HAL, RN, Mxl, BAT2, EHMT2, PRDM1, PRDM 14, or ESR.

[0014] In various configurations, the present teachings can include an oocyte prepared by a method of the present teachings.

[0015] In various configurations, the present teachings can include a swine generated from an oocyte prepared according to the present teachings.

[0016] In various embodiments, the present teachings can include a porcine metaphase II (Mil) oocyte comprising: a Cas protein or a nucleic acid encoding a Cas protein; and at least one gRNA directed to a target gene. In some configurations, the target gene can be a gene affecting a growth trait, a health trait, and a reproductive trait. In various configurations, the target gene encodes a protein involved in infection of swine or cattle by a pathogen.

[0017] In various configurations, the present teachings can include a swine generated from the oocyte prepared according to the present teachings. In various configurations, the swine does not exhibit mosaicism.

[0018] In particular, the present teachings also include a method of creating a plurality of gene-edited swine or cattle is provided. The method comprises: obtaining unfertilized oocytes from at least one heifer, cow, sow, or gilt; and introducing into the unfertilized oocytes: (a) a Cas protein or a nucleic acid encoding a Cas protein; and (b) at least one guide RNA (gRNA) directed to a target gene.

[0019] Any of the methods described herein can further comprise fertilizing the oocyte and generating an animal from the fertilized oocyte.

[0020] Porcine and bovine oocytes are also provided. The porcine or bovine oocyte can be a porcine or bovine oocyte prepared using any of the methods described herein.

[0021] Further provided is a porcine or bovine metaphase II (Mil) oocyte. The oocyte comprises a Cas protein or a nucleic acid encoding a Cas protein, and at least one gRNA directed to a target gene. [0022] Also provided are animals generated from any of the porcine or bovine oocytes described herein, and animals generated using any of the methods described herein.

[0023] Other objects and features will be in part apparent and in part pointed out hereinafter.

DETAILED DESCRIPTION

Methods for creating a plurality of gene-edited swine or cattle

[0024] The present disclosure provides a method of creating a plurality of gene-edited swine or cattle. The method comprises obtaining unfertilized oocytes from at least one heifer, cow, sow, or gilt; and introducing into the unfertilized oocytes: (a) a Cas protein or a nucleic acid encoding a Cas protein; and (b) at least one guide RNA (gRNA) directed to a target gene.

[0025] In any of the methods described herein, the unfertilized oocytes can come from a superovulated heifer, cow, sow, or gilt.

[0026] In any of the methods described herein, obtaining the unfertilized oocytes can comprise superovulating the at least one heifer, cow, sow or gilt; and harvesting unfertilized oocytes from the superovulated heifer, cow, sow, or gilt.

[0027] In any of the methods described herein, the unfertilized oocytes can be recovered from a deceased heifer, cow, sow, or gilt.

[0028] The present disclosure provides for the gene editing of unfertilized oocytes through the introduction of nucleic acid or protein molecules of a gene editing system. Gene editing can be performed using any system known in the art, for example, CRISPR/Cas, transcription activator-like effector nucleases (TALENs), prime editing, or base editing.

[0029] CRISPR/Cas editing can be accomplished using several different sets of reagents. For example, guide RNA (gRNA) can be combined with a Cas protein in vitro to form a ribonucleoprotein (RNP) complex, which can then be introduced into the oocyte. Use of RNP complexes is preferred because this reduces the half-life of the reagents and thus reduces mosaicism.

[0030] In any of the methods described herein, the at least one gRNA can comprise a single guide RNA (sgRNA). A single guide RNA (sgRNA) is a single RNA molecule that contains both a crRNA and a tracrRNA.

[0031] Any of the gRNAs can be in vitro transcribed.

[0032] Any of the gRNAs can be synthetic RNA molecules. [0033] More than one sgRNA can be administered to target different regions of the target gene, such that the double stranded breaks result in a targeted deletion. Thus, in any of the methods described herein, the at least one gRNA can comprise at least two sgRNAs. This system can be referred to as a “dual guide RNA”. This technique can use of pairs of gRNAs that result in two double stranded breaks, thus allowing for the deletion of DNA sequences: the joined ends can result in the generation of an in-frame translational stop codon across the joined ends; when the cut sites of two guides are repaired by NHEJ in an end-to-end manner, this new DNA sequence, when transcribed into mRNA and translated into protein could terminate the production of the protein encoded by the target gene.

[0034] Any of the methods described herein can comprise introducing the Cas protein into the oocyte.

[0035] As an alternative or in addition to use of a Cas protein, a nucleic acid encoding a Cas protein (e.g., an mRNA encoding a Cas protein) can be introduced into the oocyte. The nucleic acid encoding the Cas protein and the gRNA can be provided in a single vector with a Cas expression cassette and an sgRNA transcription cassette or in individual DNA vectors. [0036] The term “Cas” refers to “CRISPR-associated protein.” Cas proteins include but are not limited to Cas6 family member proteins, Cas9 family member proteins, and or a Csy4. Skilled artisans will recognize that other Cas variants are suitable for use in the present teachings. Protocols for each of the methods are known in the art. The methods used in the examples are discussed infra.

[0037] Thus, in any of the methods described herein, the Cas protein can comprise a Cas6 protein, a Cas9 protein, or a Csy4 protein. For example, the Cas protein can comprise a Cas9 protein.

[0038] As used herein, the term “directed to a target gene” means that the at least one gRNA includes a sequence that is complementary to and base pairs with a gene present in the unfertilized oocyte.

[0039] Superovulation of livestock is routinely used to increase the number of oocytes that are matured and released from the ovaries of a female animal. Methods for superovulation of heifers, cows, sows, and gilts are known in the art.

[0040] As described further herein below, following superovulation and prior to harvesting of the oocytes, the heifer, cow, sow, or gilt can be inseminated (e.g., by artificial insemination). While this procedure will result in many oocytes being fertilized and becoming zygotes, other oocytes will remain unfertilized. If the fertilization is a part of a routine fertilization, then the unfertilized oocytes might otherwise be discarded, and the method of the present teachings allows them to be utilized instead.

[0041] Following superovulation, the unfertilized oocytes (and zygotes, if present) are harvested from the heifer, cow, sow, or gilt. Several methods for harvesting unfertilized oocytes and zygotes are known in the art. These include surgical and non-surgical methods.

In pigs and cattle, once a female ovulates, the oocytes travel through the oviduct where they are normally fertilized prior to traveling into the uterus to establish a pregnancy. Thus, in pigs, flushing refers to collecting the oocytes from the female reproductive tract, specifically the oviduct. In pigs, oocytes can also be collected or aspirated from the ovary. In cattle, flushing refers to collecting the oocytes from the female reproductive tract, specifically the oviduct. In cattle, oocytes can also be collected from the ovary by a process called ovum pick up (OPU). Methods of performing flushing and ovum pick-up (OPU) are known in the art.

[0042] Where a gilt or sow is used in the methods of the present invention, harvesting the unfertilized oocytes can comprise flushing the unfertilized oocytes from the oviduct of the gilt or sow.

[0043] Where a heifer or cow is used in the methods of the present invention, harvesting the unfertilized oocytes can comprise flushing the unfertilized oocytes from the oviduct of the heifer or cow.

[0044] In any of the methods described herein, the method can further comprise maturing the unfertilized oocytes to metaphase II (Mil). In most species, the total number of eggs (oocytes) ever to be produced are present in the newborn female. These eggs are initially arrested at the diplotene stage of meiosis I in the fetus. The diplotene stage is the stage of prophase where the replicated homologous chromosomes have paired up and crossing over has occurred, but the spindle has not yet formed to progress to metaphase and separate the chromosomes. The eggs remain in the diplotene stage until after puberty, and then mature prior to ovulation. Luteinizing hormone (LH) concentration surges the day before ovulation, which stimulates the resumption of meiosis 1. At this stage, the first meiotic division occurs, separating the replicated homologous chromosomes to form two daughter cells. One cell becomes the secondary oocyte the other cell forms the first polar body. The secondary oocyte then commences to meiosis 2 which arrests at metaphase (Mil) and will not continue without fertilization. At fertilization, the sister chromatids comprising the replicated chromosomes separate and two daughter cells are formed: a fertilized oocyte with two pronuclei (one from the oocyte and one from the sperm) and a second polar body. The two pronuclei of the oocyte fuse to form a diploid zygote.

[0045] Without being limited by theory, injecting gene editing reagents into Mil oocytes allows genome editing to start occurring prior to fertilization. Because the reagents are injected at this early stage, the CRISPR-Cas9 components start to degrade before S-phase (DNA replication phase). The absence or reduction of these reagents during the DNA replication phase leads to reduced gene editing after the DNA has replicated, and therefore results in reduced mosaicism.

[0046] The maturing step is done following harvesting of the unfertilized oocytes and prior to introduction of the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA. Methods of maturing oocytes are known in the art.

[0047] In any of the methods described herein, the method can further comprise incubating the oocyte for a period of time

[0048] Any of the methods described herein can also further comprise fertilizing the oocyte.

[0049] For example, the oocyte can be fertilized immediately after introduction of the CRISPR/Cas reagents.

[0050] Where the methods comprise incubating the oocyte, the incubating step is preferably performed prior to fertilization. For example, the oocyte can be fertilized 30 minutes after introduction of the CRISPR/Cas reagents. In cattle, the oocyte can be fertilized up to 8 hours after introduction of the CRISPR/Cas reagents. Fertilization can occur 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours after introduction of the CRISPR/Cas reagents. However, optimum results are achieved at 30 minutes to 4 hours prior to fertilization. In swine, the oocyte can be fertilized up to 5 hours after the introduction of the CRISPR/CAS reagents. Fertilization can occur 0.5, 1, 2, 3, 4, or 5 hours after introduction of the CRISPR/Cas reagents. Optimum results are achieved 30 minutes to 2 hours prior to fertilization.

[0051] Thus, for example, the oocyte can be incubated for up to 0.5 hours (30 minutes) prior to fertilization, for up to 1 hour prior to fertilization, for up to 2 hours prior to fertilization, for up to 3 hours prior to fertilization, for up to 4 hours prior to fertilization, or for up to 5 hours prior to fertilization. The oocyte is suitably incubated for 30 minutes to 2 hours prior to fertilization (e.g., for 1 hour prior to fertilization).

[0052] Methods of in vitro fertilization and oocyte maturation/incubation are known in the art. [0053] The most common method for introducing CRISPR/Cas into an oocyte or zygote is via injection. Recent publications suggest that electroporation can also be used (Miao, et al., Biol. Reprod., 2019. 101, 177-187, which is hereby incorporated by reference in its entirety.) [0054] Thus, in any of the methods described herein, introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the oocyte can comprise injecting the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the oocyte.

[0055] Alternatively, or in addition, introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the oocyte can comprise electroporating the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the oocyte.

[0056] The targeted DNA sequence can be any coding or non-coding region comprising the appropriate PAM sequence for the CAS protein. The target gene can encode a protein involved in infection of swine or cattle by a pathogen. For example, the target gene can encode a protein essential for infection of swine or bovine by the pathogen. The target gene can encode a cell-surface receptor, soluble receptor, or intracellular receptor for the pathogen. [0057] The pathogen can comprise, for example, a bacterium or a virus.

[0058] Alternatively, or in addition, the target gene can encode a protein involved in the health, welfare, growth, reproduction, or wellbeing of the animal. Alternatively, or in addition, the target gene can encode a protein involved in production of milk or meat (via expansion of the animal’s muscle mass). Exemplary bovine traits include polled (lack of horns), sterility or fertility, milk production, growth (which increases meat production), fat production, conception rates, stillborn rates, calving ease, or content of produced milk such as the amount of protein or the amount of fat. Further bovine traits can include backfat thickness, intramuscular fat, ultrasound loin muscle area, loin muscle area and intramuscular fat content, chest circumference, withers height, body length, hip height, rump length, and heart girth. Further traits include high altitude adaptation and response to hypoxia (DCAF8, PPP1R12A, SLC16A3, UCP2, UCP3, TIGAR), cold acclimation (AQP3, AQP7, HSPB8), body size and stature (PLAG1, KCNA6, NDUFA9, AKAP3, C5H12orf4, RAD51AP1,

FGF6, TIGAR, CCND2, CSMD3), resistance to disease and bacterial infection (CHI3L2, GBP6, PPFIBPl, REP 15, CYP4F2, TIGD2, PYURF, SLC10A2, FCHSD2, ARHGEF17, RELT, PRDM2, KDM5B), reproduction (PPP1R12A, ZFP36L2, CSPP1), milk yield and components (NPCILI, NUDCD3, ACSS1, FCHSD2), growth and feed efficiency (TMEM68, TGS1, LYN, XKR4, FOXA2, GBP2, GBP5, FGD6), and polled phenotype (URB1, EVA1C). Exemplary target genes can include PRLR, NANOS2, APAF1, SMC2, GART, TFB1M, SIRT1, SIRT2, LPL, CRTC2, SIX4, UCP2, UCP3, URB1, EVA1C, TMEM68, TGS1, LYN, XKR4, FOXA2, GBP2, GBP5, FGD6, NPC1L1, NUDCD3, ACSS1, FCHSD2, PPP1R12A, ZFP36L2, CSPP1, CHI3L2, GBP6, PPFffiPl, REP 15, CYP4F2, TIGD2, PYURF, SLC10A2, FCHSD2, ARHGEF17, RELT, PRDM2, KDM5B, PLAG1, KCNA6, NDUFA9, AKAP3, C5H12orf4, RAD51AP1, FGF6, TIGAR, CCND2, CSMD3, AQP3, AQP7, HSPB8, DCAF8, PPP1R12A, SLC16A3, TIGAR and ZBTB. Exemplary porcine traits include meat production traits such as growth rate, backfat depth, muscle pH, purge loss, muscle color, firmness, marbling scores, intramuscular fat percentage, tenderness, average daily gain, average daily feed intake, feed efficiency, back fat thickness, loin muscle area, and lean percentage. Exemplary health traits include the absence of undesirable physical abnormalities or defects (like scrotal ruptures), improvement of feet and leg soundness, resistance to specific diseases or disease organisms, or general resistance to pathogens. Further health traits can include melanotic skin tumors, dermatosis vegetans, abnormal mamae, shortened vertebral column, kinky tail, rudimentary tail, hairlessness, woolly hair, hydrocephalus, tassels, legless, three-legged, syndactyly, polydactyly, pulawska factor, heterochromia iridis, congenital tremor a iii, congenital tremor a iv, congenital ataxia, hind leg paralysis, bentleg, thickleg, malignant hyperthermia, hemophilia (von Willebrand's disease), leukemia, hemolytic disease, edema, acute respiratory distress ("barker"), rickets, renal hypoplasia, renal cysts, uterus aplasia, porcine stress syndrome (pss), halothane (hal), dipped shoulder (humpy back, kinky back, kyphosis), hyperostosis, mammary hypoplasia, undeveloped udder, and epitheliogenesis imperfecta. Exemplary target sequences include ANP32, ANPEP, TMPRSS1, TMPRSS2, NANOS2, CD163, Melanocortin-4 receptor (MC4R), HMGA, IGF2, E.coli F4ab/ac, HAL, RN, Mxl, BAT2, EHMT2, ESR.

[0059] Persons skilled in the art will recognize that editing a gene implicated in infection by a pathogen so that the protein encoded by the gene can no longer interact with pathogen proteins will inhibit or prevent the pathogen from infecting the host cell. Thus, editing a porcine or bovine gene involved in infection of swine or cattle by a pathogen can render the animal immune or resistant to infection by the pathogen. For example, but without limitation, editing a cell-surface receptor so that it can no longer bind to a virus can prevent internalization of the virus into the cell, thus preventing the virus from infecting the cell. [0060] Generating an animal from the edited oocyte, once fertilized, can comprise implanting the fertilized oocyte into the reproductive tract of a surrogate swine, heifer, or cow, wherein gestation and term delivery results in an animal having a modification in one or both alleles of the target gene. In some configurations, gestation and term delivery results in an animal having a modification in one allele of the target gene.

[0061] Where gestation and term delivery results in an animal having a modification in only a single allele of a target gene, homozygous animals having the modification in both alleles of the target gene can be generated by breeding. For example, the method can further comprise mating a female animal produced by any of the methods described herein with a male animal produced by any of the methods described herein to produce offspring having the modification in both alleles of the target gene.

[0062] In any of the methods described herein, the oocyte can be a porcine oocyte.

[0063] In any of the methods described herein, the oocyte can be a bovine oocyte.

Porcine and bovine oocytes and animals

[0064] The present disclosure also provides porcine and bovine oocytes, as well as animals generated from the oocytes and/or generated using the methods described herein. [0065] For example, a porcine oocyte prepared by any of the methods described herein is provided.

[0066] A bovine oocyte prepared by any of the methods described herein is also provided. [0067] A further porcine or bovine oocyte is provided. The oocyte is a metaphase II (Mil) oocyte and comprises: a Cas protein or a nucleic acid encoding a Cas protein and at least one gRNA directed to a target gene.

[0068] The target gene can encode a protein involved in infection of swine or cattle by a pathogen. For example, the target gene can encode a protein essential for infection of swine or a bovine by the pathogen. The target gene can encode a cell-surface receptor, soluble receptor, or intracellular receptor for the pathogen. The pathogen can comprise a bacterium or a virus.

[0069] Alternatively, or in addition, the target gene can encode a protein involved in a trait for growth, health or welfare, or improved production of milk or meat.

[0070] The oocyte can comprise the Cas protein. The Cas protein can comprise any of the Cas proteins described herein (e.g., Cas9) or another Cas protein known in the art.

[0071] Animals generated from the oocytes and/or generated using the methods described herein are also provided.

[0072] The animal can be an animal generated from any of the oocytes described herein.

[0073] Alternatively or in addition, the animal can be an animal generated using any of the methods described herein.

[0074] The animal preferably does not exhibit mosaicism. [0075] The animal can exhibit increased resistance to infection by a pathogen as compared to a wild-type animal (for example, where the target gene encodes a protein involved in infection of swine or cattle by a pathogen).

[0076] The animal can be selected from the group consisting of a piglet, a shoat, a gilt, a sow, a barrow and a boar.

[0077] The animal can be selected from the group consisting of a calf, a heifer, a cow, a steer, or a bull.

[0078] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles “a”, “an”, “the”, and “said” are intended to mean that there are one or more of the elements.

[0079] The terms “comprising”, “including”, and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0080] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.

EXAMPLES

[0081] The following non-limiting examples are provided to further illustrate the present invention.

[0082] Methods

Ribonucleoprotein (RNP) Complex Preparation

[0083] sgRNA at a concentration of 1.5 pg/mΐ was incubated at 95°C for 2 minutes then cooled to room temperature. 1 mΐ of 10 mg/ml Cas9 protein and 1.41 mΐ of water were combined on ice. 4.56 mΐ of the denatured sgRNA was then added and the mixture incubated at room temperature for 10 minutes. For experiments using two sgRNAs, each sgRNA complex was prepared separately and then mixed at a 1:1 ratio. The RNP complexes thus formed were diluted to a working concentration of 50.2 ng/mΐ of Cas9 and 17.2 ng/mΐ of each sgRNA.

In vitro Oocyte Maturation

[0084] Porcine oocytes were matured for 20-22 hours in Maturation I medium (Applied Reproductive Technology, ART, Madison, WI), then 20-22 hours in Maturation II medium (ART). In general, Mil oocytes were ready for injection at 42 hours. Oocytes were denuded in hyaluronidase with pipetting. The denuded oocytes were washed twice. For experimental samples, the denuded Mil oocytes were then injected with the RNP complexes according to standard protocols. 25 oocytes were then aliquoted per 50 mΐ fertilization drop.

[0085] Bovine oocytes are matured for 20-22 hours in maturation medium (ART,

Madison, WI). Mil oocytes are ready for injection at 20 hours. The oocytes are denuded in a medium containing hyaluronidase with ether pipetting or vortexing. The denuded oocytes are washed twice and injected with the RNP according to standard protocols. 25 oocytes were then aliquoted per 50 mΐ fertilization drop.

In vitro Fertilization

[0086] 500 mΐ of freshly extended porcine semen was placed in a centrifuge tube and spun at 400 x g for 5 minutes. The supernatant was discarded and the sperm washed in 500 mΐ of TL HEPES, spun at 500 x g for 5 minutes, and then resuspended in equilibrated fertilization medium to a total volume of 500 mΐ. 0.6ul of sperm (1 x 10⁴ sperm/ml) was added to each 50 mΐ fertilization drop, and then sperm and oocytes were co-incubated for 3.5-4 hours.

[0087] Frozen thawed bovine semen is also used for fertilization. A semen straw is thawed at 37°C for 1 minute. Semen is layered on top of 80% BOVIPURE™ (Nidacom, Gothenburg, Sweden) and centrifuged at 500 x g for 10 minutes. The supernatant is removed, semen is washed with TL-HEPES and centrifuged 300 x g for 5 minutes. Semen is then counted and added to 500 mΐ fertilization medium with oocytes for a final concentration of 1 x 10⁶ sperms/ml. These are incubated for 18 hours.

[0088] For control samples, zygotes are injected with the RNP complexes according to standard methods. Then the zygotes are washed with TL-HEPES and 25 zygotes are added to each 50 mΐ drop of culture medium.

[0089] The genotype of blastocysts is evaluated on day 7 by Illumina sequencing.

Example 1

[0090] This example illustrates comparative experiments between control zygote injections, and a time course experiment of incubation periods between oocyte injections and fertilization.

[0091] Pig oocytes obtained from a slaughterhouse were prepared as described above. RNPs containing sgRNA were prepared as above. Injections of oocytes with the RNPs were performed and fertilization occurred immediately (0 hours), 0.5 hour, or 1 hour after the RNP injection. As a control, zygotes were injected 5 hours after fertilization. Pre-implantation results are shown in Table 1.

Table 1

In creating edited blastocysts, the results presented in Table 1 show that the injection of Mil oocytes is at least as efficient as injection of fertilized zygotes (5h post IVF).

Example 2

[0092] This example illustrates comparative experiments between oocyte and zygote injection of RNP complexes using two sgRNAs to create a desired deletion.

[0093] A 1 : 1 mixture of two sgRNA RNP complexes was prepared as described above. Porcine oocytes were injected 0 (Mil Ohr) and 1 hour (Mlllhr) prior to fertilization. Zygotes were injected 5 hours after fertilization. Pre-implantation results are shown in Table 2.

Table 2

[0094] These data indicate that injecting oocytes 1 hour prior to fertilization produces a higher frequency of the desired edit than the other treatments shown.

Example 3

[0095] This example illustrates improved efficiency of gene editing in Mil oocytes. [0096] Blastocysts as prepared in Example 1 are implanted in surrogate females and allowed to gestate until birth. Animals are then genotyped in multiple tissues. It is expected that as a group, the animals injected as oocytes will have a significantly lower incidence of mosaicism than the group of animals that are injected as zygotes.

Example 4

[0097] This example illustrates improved efficiency of desired edit in Mil oocytes.

[0098] Four cell stage embryos as prepared in Example 2 are implanted in surrogate females and allowed to gestate until birth. Animals are genotyped in multiple tissues. It is expected that as a group the animals injected as oocytes will have a significantly lower incidence of mosaicism than the group of animals that are injected as zygotes.

Example 5

[0099] This example illustrates the gene editing of oocytes from a bovine flush.

[0100] A heifer is superovulated by twice daily FSH administration for 5 days. Oocytes are recovered by standard oviductal flushing procedures. The unfertilized oocytes are confirmed to be at the right stage, metaphase II. RNP complexes comprising two sgRNAs are electroporated into the oocytes. The oocytes are incubated for 1.5 hours and then subjected to in vitro fertilization according to standard methods. The resulting zygotes are then implanted into recipient cows and gestated until birth. It is expected that the resulting calves will have a lower incidence of mosaicism as compared to calves gene edited as zygotes.

Example 6

[0101] This example illustrates the gene editing of oocytes from a bovine flush.

[0102] A heifer is superovulated by twice daily FSH administration for 5 days. Oocytes are recovered by standard flushing procedures. The unfertilized oocytes are confirmed to be at the right stage, metaphase II. RNP complexes comprising a single sgRNA are injected into the oocytes. The oocytes are incubated for 2 hours and then subjected to in vitro fertilization according to standard methods. Zygotes are then implanted into recipient heifers and gestated until birth. It is expected that the calves injected as oocytes will have reduced mosaicism as compared to calves injected as zygotes.

Example 7

[0103] This example illustrates gene editing of porcine oocytes.

A gilt was synchronized using 0.22% altrenogest followed by PMSG and hCG. Oocytes were flushed at the Mil stage. RNP complexes comprising two sgRNAs were injected into the oocytes. The oocytes were incubated for 30 minutes and then fertilized using standard methods of in vitro fertilization. The resulting zygotes were then implanted in recipient gilts and gestated until birth. For the piglet generated through the Mil injection protocol, genotype results are shown in Table 3.

Table 3

Example 8

[0104] This example illustrates gene editing of porcine oocytes from a commercial flush. [0105] A gilt is synchronized using 0.22% altrenogest followed by PMSG and hCG. Oocytes are flushed at the Mil stage. RNP complexes comprising a single sgRNA are electroporated into the oocytes. The oocytes are incubated for 1 hour and then fertilized using standard methods of in vitro fertilization. The resulting zygotes are then implanted in recipient gilts and gestated until birth. It is expected that piglets that are electroporated as oocytes will show significantly less mosaicism than piglets that are electroporated as zygotes. Example 9

[0106] This example illustrates maximum use of available oocytes from a heifer by using unfertilized oocytes from a round of ART for genome editing.

[0107] A heifer is superovulated by twice daily FSH administration for 5 days. Artificial insemination is performed according to standard methods. Zygotes and unfertilized oocytes are recovered by standard oviductal flushing procedures. Zygotes are separated and transferred to recipients. The unfertilized oocytes are staged, and oocytes confirmed to be at the right stage, metaphase II are selected. RNP complexes comprising two sgRNAs are electroporated into the oocytes. The oocytes are incubated for 1.5 hours and then subjected to in vitro fertilization according to standard methods. The resulting zygotes are then implanted into recipient cows and gestated until birth. It is expected that the resulting calves will have a lower incidence of mosaicism as compared to calves that are gene edited as zygotes.

Example 10 [0108] This example illustrates maximum use of porcine oocytes from a commercial flush of a gilt by using unfertilized oocytes from a round of ART for genome editing.

[0109] A gilt is synchronized using 0.22% altrenogest followed by PMSG and hCG. Artificial insemination is performed, and then zygotes and oocytes are flushed according to standard procedures. Zygotes are separated and implanted into recipient animals. The leftover unfertilized oocytes are staged, and oocytes at metaphase II are selected. RNP complexes comprising a single sgRNA are electroporated into the oocytes. The oocytes are incubated for 1.5 hours and then fertilized using standard methods of in vitro fertilization. The resulting zygotes are then implanted in recipient gilts and gestated until birth. It is expected that piglets that are electroporated as oocytes will show significantly less mosaicism than piglets that are electroporated as zygotes.

Claims

What is claimed is:

1. A method of creating gene-edited swine comprising: providing at least one unfertilized oocyte from a sow or a gilt; and introducing into the at least one unfertilized oocyte: (a) a Cas protein or a nucleic acid encoding a Cas protein; and (b) at least one guide RNA (gRNA) directed to a target gene.

2. The method according claim 1, further comprising maturing the at least one unfertilized oocyte to metaphase II (Mil) prior to introduction of the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA.

3. The method according to claim 1, further comprising fertilizing the at least one unfertilized oocyte after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA.

4. The method according to claim 3, wherein, after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA, the at least one oocyte is incubated for up to 5 hours prior to fertilization.

5. The method according to claim 3, wherein after introduction of the Cas protein or the nucleic acid encoding a Cas protein and the at least one gRNA, the at least one oocyte is incubated for 30 minutes to 2 hours prior to fertilization.

6. The method according to claim 1, wherein introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte comprises injecting the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte.

7. The method according to claim 1, wherein introducing the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte comprises electroporating the Cas protein or the nucleic acid encoding the Cas protein and the at least one gRNA into the at least one unfertilized oocyte.

8. The method according to claim 1, wherein the at least one gRNA comprises a single guide RNA (sgRNA).

9. The method according to claim 1, wherein the at least one gRNA comprises at least two sgRNAs.

10. The method according to claim 1, wherein the target gene affects a health trait, a reproductive trait, or a growth trait.

11. The method according to claim 1, wherein the target gene encodes a protein involved in infection of swine by a pathogen.

12. The method according to claim 11, wherein the target gene encodes a protein essential for infection of swine by the pathogen.

13. The method according to claim 1, wherein the target gene is selected from the group consisting of ANP32, ANPEP, TMPRSS1, TMPRSS2, NANOS2, CD163, Melanocortin-4 receptor (MC4R), HMGA, IGF2, E.coli F4ab/ac, HAL, RN, Mxl, BAT2, EHMT2, PRDMl, PRDM14, and ESR.

14. An oocyte prepared by a method according to claim 1.

15. A swine generated from an oocyte according to claim 14.

16. A porcine metaphase II (Mil) oocyte comprising: a Cas protein or a nucleic acid encoding a Cas protein; and at least one gRNA directed to a target gene.

17. The oocyte according claim 16, wherein the target gene is selected from the group consisting of a gene affecting a growth trait, a health trait, and a reproductive trait.

18. The oocyte according to claim 16, wherein the target gene encodes a protein involved in infection of swine by a pathogen.

19. A swine generated from the oocyte according to claim 16.

20. The swine according to claim 16, wherein the animal does not exhibit mosaicism.