WO2024097603A2 - Method of creating gene edited animals comprising a wild type allele - Google Patents

Abstract

Provided herein are methods of producing heterozygous edited animals by injecting zygotes with low amounts of ribonucleoprotein (RNP) complex. The data cited herein show that more heterozygous animals are obtained from zygotes injected with 100,000 – 450,000, or approximately 111,250 copies of an RNP complex. The methods disclosed herein are particularly useful for editing of bovine or porcine zygotes. Heterozygous animals are preferred when homozygous embryos fail to develop, or when homozygous animals are sterile, therefore making it difficult to breed the edit into a line of animals.

Description

METHOD OF CREATING GENE EDITED ANIMALS COMPRISING A WILD TYPE

ALLELE

Cross Reference to Related Application

[0001] This application claims the benefit of and priority to US Provisional application 63/382,053, filed on November 2, 2022. This application is incorporated by reference in its entirety.

Field

[0002] This application pertains the field of gene editing.

Background

[0003] The CRISPR-CAS system is a robust method of creating precise changes in an animal’s genome. In conventional methods, editing can occur on both chromosomes, often separate edits that can be the desired edit. Because the half-life of the reagents is longer than the division time of the cells, chimeric animals can result. While having two targeted edits can be advantageous, deactivation of both alleles in essential genes for survival or fertility can impede creation of gene edited lines.

[0004] Methods of performing gene editing that results in only one of two alleles being edited are needed.

Summary'

[0005] The methods of the present teachings allow for editing a single copy of a gene in a cell, so that heterozygous animals can be produced when an allele is homozygous lethal or homozygous sterile. In some embodiments, a method of creating a gene edited animal with at least one wild type allele can comprise: introducing into an isolated animal cell 100,000 - 450,000 copies of a ribonucleoprotein (RNP) complex comprising at least one guide RNA (gRNA) and a Cas9 protein; producing an animal from isolated animal cell. In some configurations, about 100,000 copies, about 105,000 copies, about 110,000 copies, about 115,000 copies, about 120,000 copies, about 125,000 copies, about 130,000 copies, about 135,000 copies, about 140,000 copies, about 145,000 copies, 150,000 copies, about 155,000 copies, about 160,000 copies, about 165,000 copies, about 170,000 copies, about 175,000 copies, about 180,000 copies, about 185,000 copies, about 190,000 copies, about 195,000 copies, about 200,000 copies, about 205,000 copies, about 210,000 copies, about 215,000 copies, about 220,000 copies, about 225,000 copies, about 230,000 copies, about 235,000 copies, about 240,000 copies, about 245,000 copies, about 250,000 copies, about 255,000 copies, about 260,000 copies, about 265,000 copies, about 270.000 copies, about 275,000 copies, about 280,000 copies, about 285,000 copies, about 290,000 copies, about 295,000 copies, about 300,000 copies, about 305,000 copies, about 310,000 copies, about 315,000 copies, about 320,000 copies, about 325,000 copies, about 330,000 copies, about 335,000 copies, about 340,000 copies, about 345,000 copies, 350,000 copies, about 355,000 copies, about 340,000 copies, about 345,000 copies, about 350,000 copies, about 355,000 copies, about 360,000 copies, about 365,000 copies, about 370,000 copies, about 375,000 copies, about 380,000 copies, about 385,000 copies, about 390,000 copies, about 395,000 copies, about 400,000 copies, about 405,000 copies, about 410,000 copies, about 415,000 copies, about 420,000 copies, about 425,000 copies, about 430,000 copies, about 435,000 copies, about 440,000 copies, about 445,000 copies, or 450,000 copies of the RNP complex can be introduced into the isolated animal cell. In various configurations, 110,000-440,000 copies, 120,000-430,000 copies. 100,000-250,000 copies. 110,000-220,000 copies, or 120,000- 250,000 copies of the RNP complex can be introduced into the isolated animal cell.

[0006] In various configurations, the isolated animal cell can be an oocyte and the producing can comprise fertilizing the oocyte. In some configurations, the producing can further comprise implanting the fertilized oocyte into a surrogate mother. In various configurations, the isolated animal cell can be a zygote and the producing can comprise implanting the zygote into a surrogate mother. In various configurations, the isolated animal cell can be a fibroblast cell or an embryonic stem cell. In various configurations, the producing can comprise nuclear transfer.

[0007] In various configurations, the introducing can comprise introducing 200,000 - 400,000 copies of an RNP complex into an isolated animal cell. In some configurations, the introducing comprises introducing about 110,000 copies of an RNP complex into an isolated animal cell. In various configurations, the introducing can comprise introducing about 220,000 copies of an RNP complex into an isolated animal cell. In various configurations, the introducing can comprise introducing about 222,500 copies of an RNP complex into an isolated animal cell. In various configurations, the introducing can comprise introducing about 111,250 copies of an RNP complex into an isolated animal cell.

[0008] In various configurations, the at least one gRNA is a pair of gRNAs. In various configurations, the at least one gRNA can create an edit that is deleterious to the gene edited animal if the edit is homozygous. In various configurations, the at least one gRNA can create an edit that is homozygous lethal. In various configurations, the at least one gRNA can create an edit that is homozygous sterile. In various configurations, the at least one gRNA can create an edit that results in an allele that creates a desirable phenotype when it is combined with a different allele. [0009] In various configurations, the animal can be a mammal. In some configurations, the animal can be a livestock animal. In some configurations, the animal can be an ungulate. In some configurations, the animal can be a porcine animal. In some configurations, the animal can be a pig. In various configurations, the animal can be a bovine animal. In some configurations, the animal can be a Bos taurus.

[0010] In various configurations, the gene edited animal can comprise at least some cells that have one wild type allele and one edited allele. In various configurations, the isolated animal cell is a zygote and the producing comprises implanting the zygote into a surrogate mother.

[0011] In various configurations, the method can further comprise breeding the animal to a wild type animal.

[0012] In various configurations, the disclosure provides for a pig produced by the instant methods.

[0013] In various configurations, the disclosure provides for a Bos taurus produced by the instant methods.

[0014] In various configurations, the introducing of the 100,000 - 450,000 copies of the RNP complex comprising the at least one gRNA and the Cas9 protein causes a gene edit in the isolated animal cell.

[0015] In various embodiments, the instant disclosure includes a method of creating a gene edited animal with a wild type allele that can comprise: introducing into a zygote about 1 1 1,250 copies of a ribonucleoprotein (RNP) complex comprising at least one guide RNA (gRNA) and a Cas9 protein; and implanting the zygote into a surrogate mother thereby obtaining a gene edited animal comprising at least some cells with one copy of an edited gene and one copy of a wild t pe gene. In some configurations, the zygote can be a bovine zygote or a porcine zygote.

[0016] In various configurations, the disclosure provides for a pig produced by the instant methods.

[0017] In various configurations, the disclosure provides for a Bos taurus produced by the instant methods.

[0018] In various configurations, the introducing of the 100,000 - 450,000 copies of the RNP complex comprising the at least one gRNA and the Cas9 protein causes a gene edit in the isolated animal cell.

Detailed Description [0019] In general, the methods of the present teachings encompass introducing ribonucleic acid complexes comprising guide RNAs (gRNAs) and Cas9 proteins into an isolated mammalian cell that allow for the preservation of one wild type allele of the edited cell. More specifically, these methods comprise using reduced amounts of gRNA/Cas9 reagents compared to a regular CRISPR reaction — a reduction of about 50% to about 88%; in other terms, the mix of ribonucleoprotein complexes (RNPs) is diluted to a concentration from 1:2 to 1:8.

[0020] As used herein, the term “livestock animal” includes any animals traditionally raised in livestock farming, for example ungulates such as artiodactyls, avian animals such as chickens, turkeys, ducks, geese, guinea fowl, or squabs, or equine animals such as horses or donkeys. Ungulates include, but are not limited to porcine animals such as pigs, bovine animals such as beef or daily' cattle or buffalo, ovine animals such as sheep, caprine animals such as goats, camels, llamas, alpacas, and deer. The term “livestock animal” does not include rats, mice, or other rodents.

[0021] As used herein, the term "ungulate" refers to a diverse group of large mammals that includes but is not limited to equines, bovines or cattle, porcines or swine, goats, buffalo, sheep, giraffes, camels, deer, and hippopotamuses. Most terrestrial ungulates use the tips of their toes, usually hoofed, to sustain their whole body weight while moving. In some embodiments, the term means, roughly, "being hoofed" or "hoofed animal."

[0022] Studies of the type II CRISPR/Cas system of Streptococcus pyogenes have shown that three components form a RNA/protein complex and together are sufficient for sequencespecific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek, M. et al., Science, 2012, 337, 816-821).

[0023] It was further demonstrated that a synthetic chimeric gRNA composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary' to the crRNA in vitro. It was also demonstrated that transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double-stranded breaks (DSBs) in a variety of different species.

[0024] Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III. where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu, P.D., et al., Cell, 2014, 157, 1262- 1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA that combines a crRNA and a tracrRNA into a single molecule. The amino acid sequence of a Cas9 protein described herein, as well as certain other Cas proteins herein, may be derived ixom a Streptococcus (e.g., S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae, S. parasanguinis , S. oralis, S. salivarius, S. macacae, S. dysgalactiae, S. anginosus, S. constellatus , S.pseudoporcinus, S. mutans), Listeria (e.g., L. innocud), Spiroplasma (e.g., S. apis, S. syrphidicola), Peptostreptococcaceae, Atopobium, Porphyromonas (e.g., P. catoniae), Prevotella (e.g., P. intermedia), Veillonella, Treponema (e.g., T socranskii, T denticola), Capnocytophaga, Finegoldia (e.g., F. magna), Coriobacteriaceae (e.g., C. bacterium), O. senella (e.g., O. profusa), Haemophilus (e.g., H. sputorum, H. pittmaniae), Pasteurella (e.g., P. bettyae), Olivibacter (e.g., O. sitiensis). Epilithonimonas (e.g., E. tenax), Mesonia (e.g. , M. mobilis). Lactobacillus (e.g., L. plantarum). Bacillus (e.g., B. cereus), Aquimarina (e.g., A. muelleri), Chryseobactenum (e.g., C. palustre), Bacteroides (e.g., B. graminisolvens), Neisseria (e.g., N. meningitidis), Francisella (e.g., F. novicida), or Flavobacterium (e.g., F. frigidarium, F. soli) species, for example. As another example, a Cas9 protein can be any of the Cas9 proteins disclosed in Chylinski, K., et al., RNA Biology, 10, 726-737) which is incorporated herein by reference.

[0025] Accordingly, the sequence of a Cas9 protein herein can comprise, for example, any of the Cas9 amino acid sequences disclosed in GenBank Accession Nos. G3ECR1 (S. thermophilus), WP_026709422, WP_027202655, WP_027318179, WP_027347504, WP_0273768I5, WP_027414302, WP_027821588, WP_027886314, WP_027963583, WP_028123848, WP_028298935, Q03J16 (A thermophilus), EGP66723, EGS38969, EGV05092, EH165578 (S. pseudoporcinus), EIC75614 (S. oralis), EID22027 ( . constellatus), EIJ69711 , EJP22331 (S. oralis), EJP26004 (S. anginosus), EJP30321, EPZ44001 (5. pyogenes), EPZ46028 (S. pyogenes), EQL78043 (S. pyogenes), EQL78548 (S. pyogenes), ERLI05H, ERL12345, ERL19088 (S. pyogenes), ESA57807 (S. pyogenes), ESA59254 (S. pyogenes), ESU85303 (5. pyogenes), ETS96804, UC75522, EGR87316 (A dysgalactiae), EGS33732, EGV01468 (S. oralis), EHJ52063 (S. macacae), EID26207 (S. oralis). EID33364. EIG27013 (A parasanguinis). EJF37476, EJO19166 (Streptococcus sp. BS35b), EJU16049, EJU32481, YP_006298249, ERF61304, ERK04546, ETJ95568 (S. agalactiae), TS89875, ETS90967 (Streptococcus sp. SR4), ETS92439, EUB27844, (Streptococcus sp. BS21), AFJ08616, EUC82735 (Streptococcus sp. CM6), EWC92088, EWC94390, EJP25691, YP_008027038, YP_008868573, AGM26527, AHK22391, AHB36273, Q927P4, G3ECR1, or Q99ZW2 (S. pyogenes), which are incorporated by reference.

[0026] In some configurations, the gRNA and Cas9 are introduced to the isolated cell as pre-complexed RNPs: with the Cas9 already bound to the gRNA. In a typical CRISPR reaction, 20 pmol of Cas9 can be mixed with 60 pmol of sgRNAs and then 20 pl can be injected into the isolated cell. In some configurations, this leads to an injection of about 1.5 amol, which equals about 890,000 copies of the RNP complex. However, the exact weight in ng or molarity of the RNPs will depend on the size of the gRNAs; skilled artisans will be able to calculate the number of copies based on the size and concentration of each gRNA-Cas9 complex. In this exemplary protocol, diluting the RNPs 1:2 prior to injection leads to an injection of about 0.75 amol, or about 445,000 copies of RNP complex. A 1 :4 dilution leads to injection of about 0.375 amol. or about 222,500 copies of the RNP complex. A 1:8 dilution leads to injection of about 0. 1875 amol, or about 111,250 copies of the RNP complex.

[0027] The isolated cell can be any cell which can be used to make a full grown animal: either through natural development, cell nuclear transfer, or other methods known in the art. In various embodiments, the isolated cell can be an oocyte, a zygote, a cultured cell such as a fibroblast cell, or an embryonic stem cell. In embodiments where the cell is an oocyte, the producing can comprise fertilizing the oocyte and then implanting the fertilized oocyte into a surrogate mother. In embodiments w here the isolated cell is a zygote, the producing can comprise implanting the zygote into a surrogate mother. In embodiments where the cell is an embryonic stem cell or a culture cell, the producing can comprise creating an embryo via nuclear transfer or embryo complementation. Alternatively, or in addition, the embryonic stem cells can be directly induced to become an embryo. Once an embryo is obtained, the embryo is implanted into a surrogate mother for gestation.

[0028] Pronuclear staged fertilized eggs can be obtained in vitro or in vivo (i.e., surgically recovered from the oviduct of donor animals). In vitro fertilized eggs can be produced as follows. For example, bovine ovaries can be collected at an abattoir, and maintained at 22- 28°C. during transport. Ovaries can be washed and isolated for follicular aspiration, and follicles ranging from 2-10 mm can be aspirated into 50 mL conical centrifuge tubes using 18-gauge needles and under vacuum. Follicular fluid and aspirated oocytes can be rinsed through pre-filters with commercial TL-HEPES (ABT 360, Pullman, WA). Oocytes surrounded by a compact cumulus mass can be selected and placed into BO-IVM medium (IVF Bioscience. Cornwall, UK) for approximately 22 hours in humidified air at 38.5° C and 5% CO2. Matured oocytes can be stripped of their cumulus cells by vortexing in 0.1% hyaluronidase for 1 minute.

[0029] Alternatively, or in addition, oocytes can be har ested from elite donors, such an elite cow or heifer, or a gilt or sow of an elite line such as a PIC™ line, via methods known in the art such as superovulation and flushing or ovum pick up.

[0030] For cattle, mature oocytes can be fertilized in 50 pl BO-IVM medium. In preparation for in vitro fertilization (IVF), frozen bull semen can be washed and resuspended in TL-HEPES (Minitube, Verona, WI) to 10 million sperm/mL. Final in vitro insemination can be performed at a final concentration of approximately 5.000 motile sperm/oocyte. depending on the bull and ejaculate. All fertilizing oocytes can be incubated at 38.5°C in 5.0% CO2 atmosphere for 16 hours.

[0031] For swine, mature oocytes can be fertilized in 500 pl Minitube PORCPRO IVF MEDIUM SYSTEM (Minitube, Verona, Wis.) in Minitube 5-well fertilization dishes. In preparation for in vitro fertilization (IVF), freshly -collected or frozen boar semen can be washed and resuspended in PORCPRO IVF Medium to 4x10’ sperm. Sperm concentrations can be analyzed by computer assisted semen analysis (SPERMVISION, Minitube, Verona, WI). Final in vitro insemination can be performed in a 10 pl volume at a final concentration of approximately 40 motile sperm/oocyte. depending on the boar. The oocytes can be incubated at 38.7° C in 5.0% CO2 atmosphere for 6 hours. Six hours post-insemination, presumptive zy gotes can be washed twice in NCSU-23 and moved to 0.5 mL of the same medium. This system can produce 20-30% blastocysts routinely across most boars with a 10- 30% polyspermic insemination rate.

[0032] Linearized nucleic acid constructs, mRNA, or protein can be injected into one of the pronuclei or into the cytoplasm. Then the injected eggs can be transferred to a recipient female (e.g., into the oviducts of a recipient female) and allowed to develop in the recipient female to produce the transgenic or gene edited animals. In particular, in vitro fertilized embryos can be centrifuged at 15,000 x g for 5 minutes to sediment lipids allowing visualization of the pronucleus. The embryos can be injected with linearized nucleic acid constructs, mRNA, or protein using an Eppendorf FEMTOJET® (Eppendorf AG, Germany) injector and can be cultured until blastocyst formation. Rates of embryo cleavage and blastocyst formation and quality can be recorded. [0033] Embry os can be transferred into uteri of synchronous or asynchronous recipients. After transfer, real-time ultrasound examination of pregnancy can be performed.

[0034] In somatic cell nuclear transfer, a gene edited cell such as an embryonic blastomere, fetal fibroblast, adult ear fibroblast, or granulosa cell that includes a nucleic acid construct described above, can be introduced into an enucleated oocyte to establish a combined cell. Oocytes can be enucleated by partial zona dissection near the polar body and then pressing out cytoplasm at the dissection area. Typically, an injection pipette with a sharp beveled tip is used to inject the transgenic or gene edited cell into an enucleated oocyte arrested at meiosis-2. In some conventions, oocytes arrested at meiosis-2 are termed eggs. After producing a bovine or porcine embry o (e.g., by fusing and activating the oocyte), the embryo is transferred to the oviducts of a recipient female, about 20 to 24 hours after activation. See, for example, Cibelli, J.B., et al., Science, 1998, 280, 1256-1258 or U.S. Pat. Nos. 6,548,741, 7,547,816, 7,989,657, or 6,211,429. For cattle, recipient females can be checked for pregnancy approximately 20-21 days after transfer of the embry os. For swine, recipient females can be checked for pregnancy approximately 28 days via ultrasound; pregnancy is also marked by a lack of return to estrus after 21 days.

[0035] Standard breeding techniques can be used to create animals that are homozygous for the inactivated gene from the initial heterozy gous founder animals. Homozy gosity⁷ may not be required, however. Gene edited cattle described herein can be bred with other cattle of interest.

[0036] Once gene edited animals have been generated, modification of an endogenous nucleic acid can be assessed using standard techniques. Initial screening can be accomplished by Southern blot analysis to determine whether or not inactivation has taken place. For a description of Southern analysis, see sections 9.37-9.52 of Sambrook et al.. 1989, Molecular Cloning, A Laboratory Manual, second edition. Cold Spring Harbor Press, Plainview; N.Y. Polymerase chain reaction (PCR) techniques also can be used in the initial screening PCR refers to a procedure or technique in which target nucleic acids are amplified. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers ty pically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. PCR is described in, for example PCR Primer: A Laboratory Manual, ed. Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. Nucleic acids also can be amplified by ligase chain reaction, strand displacement amplification, selfsustained sequence replication, or nucleic acid sequence-based amplification. See, for example, Lewis, R., Genetic Engineering News, 1992, 12,1 ; Guatelli, J.C., et al.. Proc. Natl. Acad. Sci. USA, 1990, 87, 1874-1878; and Weiss, R., Science, 1991, 254, 1292-1293. At the blastocyst stage, embryos can be individually processed for analysis by PCR, Southern hybridization, and splinkerette PCR (see, e.g., Dupuy. A. J., et al.. Proc. Natl. Acad. Sci. USA, 99. 2002, 4495-4499).

[0037] A tissue sample can be taken from an animal with a genome edited according to the disclosure herein. Tail, ear notch, or blood samples are suitable tissue types. The tissue sample can be frozen at -20°C within 1 hour of sampling to preserve integrity of the DNA in the tissue sample.

[0038] DNA can be extracted from tissue samples after proteinase K digestion in lysis buffer. Characterization can be performed on two different sequence platforms, short sequence reads using the ILLUMINA® platform (ILLUMINA®, San Diego, CA) and long sequence reads on an Oxford NANOPORE™ platform (Oxford NANOPORE™ Technologies, Oxford, UK).

[0039] For short sequence reads, two-step PCR can be used to amplify and sequence the region of interest. The first step can be a locus-specific PCR which amplifies the locus of interest from the DNA sample using a combined locus-specific primer with a vendor-specific primer. The second step can attach the sequencing index and adaptor sequences to the amplicon from the first step so that sequencing can occur.

[0040] The locus-specific primers for the first step PCR can be chosen so that they amplify' a region of less than 300 base pairs such that ILLUMINA® paired-end sequencing reads could span the amplified fragment. Multiple amplicons are preferred to provide redundancy should deletions or naturally occurring point mutations prevent primers from correctly binding. Sequence data for the amplicon can be generated using an ILLUMINA® sequencing platform (MISEQ®, ILLUMINA®, San Diego, CA). Sequence reads can be analyzed to characterize the outcome of the editing process.

[0041] For long sequence reads, two-step PCR can be used to amplify and sequence the region of interest. The first step is a locus-specific PCR which amplifies the locus of interest from the DNA sample using a combined locus-specific primer with a vendor-specific adapter. The second step PCR attaches the sequencing index to the amplicon from the first-step PCR so that the DNA is ready for preparing a sequencing library. The step 2 PCR products undergo a set of chemical reactions from a vendor kit to polish the ends of the DNA and ligate on the adapter containing the motor protein to allow access to the pores for DNA strand-based sequencing.

[0042] The locus specific primers for the first step PCR range will be designed to amplify different regions of the gene of interest and will amplify' regions different in length. Normalized DNA can be mixed with vendor supplied loading buffer and loaded onto the NANOPORE™ flowcell.

[0043] Long sequence reads, while having lower per base accuracy than short reads, are very useful for observing the long-range context of the sequence around the target site. Founder Animals, Traits, and Reproduction

[0044] Founder animals may be produced by cloning and other methods described herein. The founders can be homozygous for a genetic modification, as in the case where a zygote or a primary cell undergoes a homozygous modification. Similarly, founders can also be made that are heterozy gous. Founders can be mosaic for a modification, as may happen when vectors are introduced into one of a plurality’ of cells in an embryo, typically at a blastocyst stage or if the reagents are injected into a zygote but degrade at a slower rate than the cell divides. Progeny' of mosaic animals may be tested to identify progeny that carry the gene edit.

[0045] In addition to monitoring traits, artisans can look at the genetic background of the animal as a whole. Genomic edits such as those of the present teachings may be made in elite genetic backgrounds.

[0046] For example, when the animal is a porcine animal, the isolated cell may come from a homogenous line of elite porcine germplasm. For example, but without limitation, Elite PIC™ (Pig Improvement Company. Limited, Basingstoke, UK) lines 2, 3, 15, 19, 27, 62 and 65 are lines selected for superior commercial phenotypes. Fibroblast cell lines can be grown from collagenase treated ear notch samples extracted from the animals from these cell lines using methods known in the art.

[0047] Other potential porcine lines include lines can be PIC™ Line 15, PIC™ Line 17. PIC™ Line 27. PIC™ Line 65, PIC™ Line 14, PIC™ Line 62, P1C337, P1C800. PIC280, PIC327, PIC408, PIC™ 399, PIC410, PIC415, PIC359, PIC380, PIC837, PIC260, PIC265, PIC210, PIC™ Line 2, PIC™ Line 3, PIC™ Line 4, PIC™ Line 5, PIC™ Line 1 , PIC™ Line 19, PIC™ Line 92, PIC95, PIC™ CAMBOROUGH® (Pig Improvement Company, Limited, Basingstoke, UK), PIC 1070, PIC™ CAMBOROUGH® 40, PIC™ CAMBOROUGH® 22, PIC 1050, PIC™ CAMBOROUGH® 29, PIC™ CAMBOROUGH® 48, or PIC™ CAMBOROUGH® x54.

[0048] In various aspects, PIC™ Line 65 is sold under the trade name PIC337. In various aspects, PIC™ line 62 is sold under the tradename PIC408. In various aspects, hybrid pigs made by crossing PIC™ lines 15 and 17 are sold under the tradenames PIC800 or PIC280. In various aspects, PIC™ Line 27 is sold under the tradename PIC327. In various aspects, hybrids created from crossing PIC™ Line 65 and PIC™ Line 62 are sold under the tradenames PIC399, PIC410, or PIC415. In various aspects, hybrids created from crossing PIC™ Line 65 and PIC™ Line 27 are sold under the tradename PIC359. In various aspects, hybrids prepared from crossing PIC™ Line 800 pigs (which is a hybrid of PIC™ Line 15 and PIC™ Line 17) to PIC™ Line 65 pigs are sold under the tradenames PIC380 or PIC837. In various aspects, PIC™ Line 14 is sold under the trade name PIC260. In various aspects, hybrids created from crossing PIC™ Line 14 and PIC™ Line 65 are sold under the tradename PIC265. In various aspects, hybrids created by crossing PIC™ Line 2 and PIC™ Line 3 are sold under the tradenames PIC210, PIC™ CAMBOROUGH®, and PIC 1050. In various aspects, hybrids of PIC™ Line 3 and PIC™ Line 92 are sold under the tradename PIC95. In various configurations, hybrids made from crossing PIC™ Line 19 and PIC™ Line 3 are sold under the tradename PIC1070. In various aspects, hybrids created by crossing PIC™ Line 18 and PIC™ Line 3 are sold under the tradename PIC™ CAMBOROUGH® 40. In various aspects, hybrids created from crossing PIC™ Line 19 and PIC 1050 (which is itself a hybrid of PIC™ lines 2 and 3) are sold under the tradename PIC™ CAMBOROUGH® 22. In various aspects, hybrids created from crossing PIC™ Line 2 and PIC 1070 (which is itself a hybrid of PIC™ lines 19 and 3) are sold under the tradename PIC™ CAMBOROUGH® 29. In various aspects, hybrids created from crossing PIC™ Line 18 and PIC 1050 (which is itself a hybrid of PIC™ lines 2 and 3) are sold under the tradename PIC™ CAMBOROUGH® 48. In various aspects, hybrids created from crossing PIC™ Line 4 and PIC™ Line 5 are sold under the tradename PIC™ CAMBOROUGH® x54

Confirmation of Edits

[0049] One useful method of detecting a desired edit is to use real-time PCR. PCR primers flanking the region of interest and a probe that specifically anneals to the region of interest can be designed. The probe can be labelled with both a fluorophore and a quencher. In the PCR reaction, the primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand of the region of interest. Because the probe is intact, the fluorophore and quencher are in close proximity and the quencher absorbs fluorescence emitted by the fluorophore. The polymerase extends from the primers and begins DNA synthesis. When the polymerase reaches the probe, the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces. This fluorescence is detected by the real time instrument. These steps are repeated for each PCR cycle and allow detection of specific products. A commercial real-time PCR kit can be used to probe various animals for the desired edit. A variety of commercial real-time PCR kits exist including, such as, but without limitation, PRIMETIME®* from IDT, TAQMAN®¹ (Roche Molecular Systems, Inc, Pleasonton, CA) from Applied Biosystems, and various kits from Qiagen and Bio-Rad. Skilled persons will recognize that any such kit can be used with the primers and methods of the present teachings to achieve like results.

EXAMPLES

[0050] The present teachings including descriptions provided in the Examples that are not intended to limit the scope of any claim or embodiment. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art. in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

[0051] This example illustrates in vivo gene editing by injection of RNPs.

[0052] For each experiment, the gRNAs were generated by in vitro transcription or chemically synthesized (synthetic sgRNA) by a commercial vendor (IDT) and complexed with IDT Alt-R spCas9 V3 in water, using 3.2 pg of Cas9 protein and 2.2 pg of gRNA in a total volume of 2.23 pl. For dual guide experiments the resulting RNP complexes were then combined 1: 1 in a total volume of 2.23 pl to generate gRNA pairs. For both single and dual guide experiments, the end result was a solution of about 20 pmol Cas9 and 60 pmol sgRNAs. As used throughout these examples, this is the lx solution, and contains about 1.5 amol, or about 890,000 copies of the RNP complex. This solution was then diluted to make other concentrations of RNP complex: a 1:2 dilution (l/2x or 0.5x), which results in injection of about 0.75 amol, or about 445,000 copies of the RNP complex, a 1 :4 dilution (l/4x or 0.25x), which results in injection of about 0.375 amol, or about 222,500 copies of RNP complex, and a 1:8 dilution (l/8x or 0.125x), which results in injection of about 0.1875 amol, or about 111,250 copies of the RNP complex. [0053] The sgRNP solution was injected into the cytoplasm of presumptive zy gotes at 16- 17 hours post-fertilization by using a single pulse from a FEMTOJET® 4i microinjector (Eppendorf; Hamburg, Germany), which released about 20 picoliters per zygote.

Microinjection was performed in TL-Hepes (ABT3 0, LLC) supplemented with 3 mg/ml BSA (Proliant) on the heated stage of an inverted microscope equipped with Narishige micromanipulators (Narishige International USA, Amityville, NY). Following injections, presumptive porcine zygotes were cultured for 7 days in PZM5 (Cosmo Bio, Co LTD, Tokyo, Japan) or a bovine in vitro culture medium in an incubator environment of 5% CO2, 5% O2, 90% N2.

Example 2

[0054] This example illustrates increasing frequency of wild type allele of a dual guide porcine editing strategy.

[0055] Guides 1 and 2 were complexed with Cas9 to form RNPs and dilutions prepared as described in Example 1. The complexed guides were then injected into porcine zygotes. The zygotes were then cultured to blastocyst stage, lysed with commercial lysate buffer and used as a template for PCR. The PCR template was then sequenced using NextGen sequencing. The frequency of the wild type allele and frequency of zygotes comprising the wild type allele and the desired edit are shown in Table 1. The dose is the concentration of RNPs relative to the amount described in Example 1.

Table 1

[0056] This example illustrates that reduced amounts of gRNA-CAS9 RNPs leads to increased wild type allele frequency in porcine zygotes.

Example 3

[0057] This example illustrates a method of increasing wild type allele frequency in bovine zygotes. [0058] 4 sets of dual guides were injected into bovine embry os as described in Example 1 at lx and l/2x. Embryos were cultured as in Example 1, and sequenced as described in Example 1. Two different repetitions were completed--the results are shown in Table 2.

[0059] The results are described in Table 2. Treatments A, B, and C refer to separate sets of dual guides with concentrations relative to the concentrations described in Example 1, in RNP complexes with IDT Alt-R spCas9 V3.

Table 2

[0060] These experiments demonstrate that lower concentrations result in an increase of animals that are heterozygous for the desired edit.

[0061] All publications cited herein are hereby incorporated by reference, each in their entirety.

Claims

What is Claimed is:

1. A method of creating a gene edited animal with at least one wild type allele comprising: introducing into an isolated animal cell 100,000 - 450,000 copies of a ribonucleoprotein (RNP) complex comprising at least one guide RNA (gRNA) and a Cas9 protein; producing an animal from the isolated animal cell.

2. The method of claim 1, wherein the isolated animal cell is an oocyte and the producing comprises fertilizing the oocyte.

3. The method of claim 2, wherein the producing further comprises implanting the fertilized oocyte into a surrogate mother.

4. The method of claim 1, wherein the isolated animal cell is a zygote and the producing comprises implanting the zygote into a surrogate mother.

5. The method of claim 1, wherein the isolated animal cell is a fibroblast cell or an embryonic stem cell.

6. The method of claim 5, wherein the producing comprises nuclear transfer.

7. The method of claim 1, wherein the introducing comprises introducing 100,000 - 250,000 copies of an RNP complex into an isolated animal cell.

8. The method of claim 1, wherein the introducing comprises introducing about 110,000 copies of an RNP complex into an isolated animal cell.

9. The method of claim 1, wherein the introducing comprises introducing about 220,000 copies of an RNP complex into an isolated animal cell.

10. The method of claim 1, wherein the introducing comprises introducing about 222,500 copies of an RNP complex into an isolated animal cell.

11. The method of claim 1, wherein the introducing comprises introducing about 111,250 copies of an RNP complex into an isolated animal cell.

12. The method of claim 1, wherein the at least one gRNA is a pair of gRNAs.

13. The method of claim 1, wherein the at least one gRNA creates an edit that is deleterious to the gene edited animal if the edit is homozy gous.

14. The method of claim 1, wherein the at least one gRNA creates an edit that is homozygous lethal.

15. The method of claim 1, wherein the at least one gRNA creates an edit that is homozygous sterile.

1 . The method of claim 1, wherein the at least one gRNA creates an edit that results in an allele that creates a desirable phenotype when it is combined with a different allele.

17. The method of claim 1, wherein the gene edited animal is a mammal.

18. The method of claim 1, wherein the gene edited animal is a porcine animal.

19. The method of claim 1, wherein the gene edited animal is a pig.

20. The method of claim 1, wherein the gene edited animal is a bovine animal.

21. The method of claim 1, wherein the gene edited animal is a Bos taurus.

22. The method of claim 1, wherein the gene edited animal comprises at least some cells that have one wild type allele and one edited allele.

23. The method of claim 22, wherein the isolated animal cell is a zy gote and the producing comprises implanting the zygote into a surrogate mother.

24. The method of claim 1, further comprising breeding the animal to a wild type animal.

25. A pig produced by the method of claim 1.

26. A Bos taurus produced by the method of claim 1.

27. The method of claim 1, wherein the introducing of the 100,000 - 450,000 copies of the RNP complex comprising the at least one gRNA and the Cas9 protein causes a gene edit in the isolated animal cell.

28. A method of creating a gene edited animal w ith a wild type allele comprising: introducing into a zygote about 111,250 copies of a ribonucleoprotein (RNP) complex comprising at least one guide RNA (gRNA) and a Cas9 protein; and implanting the zy gote into a surrogate mother thereby obtaining a gene edited animal comprising at least some cells with one copy of an edited gene and one copy of a wild type gene.

29. The method of claim 28, wherein the zygote is a bovine zy gote or a porcine zygote.

30. A pig produced by the method of claim 28.

31. A Bos laurus produced by the method of claim 28.

32. The method of claim 28, wherein the introducing of the about 111,250 copies of the RNP complex comprising the at least one gRNA and the Cas9 protein causes a gene edit in the isolated animal cell.