title: Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.
abstract: Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Whitworth
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Kiho
authors.initials: K
authors.lastname: Lee
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Benne
authors.affiliation: National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Beaton
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Lee D
authors.initials: LD
authors.lastname: Spate
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Murphy
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Samuel
authors.affiliation: National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Jiude
authors.initials: J
authors.lastname: Mao
authors.affiliation: National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Chad
authors.initials: C
authors.lastname: O'Gorman
authors.affiliation: National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Walters
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Clifton N
authors.initials: CN
authors.lastname: Murphy
authors.affiliation: University of Florida, Gainesville, Florida.
authors.forename: John
authors.initials: J
authors.lastname: Driver
authors.affiliation: Genus, plc, DeForest, Wisconsin.
authors.forename: Alan
authors.initials: A
authors.lastname: Mileham
authors.affiliation: Genus, plc, DeForest, Wisconsin.
authors.forename: David
authors.initials: D
authors.lastname: McLaren
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri.
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
authors.affiliation: Division of Animal Science, University of Missouri-Columbia, Columbia, Missouri National Swine Resource and Research Center, University of Missouri-Columbia, Columbia, Missouri pratherr@missouri.edu.
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000502:physiology*; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D055610:Antigens, CD1d / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000378:metabolism; D001755:Blastocyst / Q000502:physiology*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D046149:Embryo Culture Techniques / Q000662:veterinary; D004624:Embryo Transfer / Q000662:veterinary; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D017353:Gene Deletion; D005818:Genetic Engineering / Q000009:adverse effects / Q000379:methods / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008297:Male; D009154:Mutation; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D009865:Oocytes / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034421:Sus scrofa / Q000235:genetics / Q000502:physiology*; D019076:Transgenes; D000098902:CD163 Antigen
chemical_list: D015703:Antigens, CD; D055610:Antigens, CD1d; D015214:Antigens, Differentiation, Myelomonocytic; D000098902:CD163 Antigen; D011956:Receptors, Cell Surface; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: CRISPR/Cas9; blastocyst; embryo; genetic engineering; porcine/pig; somatic cell nuclear transfer
references.citation: Curr Opin Microbiol. 2011 Jun;14(3):321-7
references.pmid: 21531607
references.citation: Sci Rep. 2013;3:1981
references.pmid: 23760311
references.citation: Nature. 2007 Jul 5;448(7149):44-9
references.pmid: 17581592
references.citation: Genesis. 2002 Nov;34(3):203-7
references.pmid: 12395385
references.citation: Trends Biotechnol. 2013 Jul;31(7):397-405
references.pmid: 23664777
references.citation: Mol Reprod Dev. 2013 Feb;80(2):145-54
references.pmid: 23239239
references.citation: Sci Rep. 2013 Oct 10;3:2847
references.pmid: 24108318
references.citation: Mol Reprod Dev. 2011 Jan;78(1):2
references.pmid: 21268178
references.citation: Cell Res. 2011 Jun;21(6):979-82
references.pmid: 21502977
references.citation: Cell Reprogram. 2010 Feb;12(1):75-83
references.pmid: 20132015
references.citation: Biol Reprod. 2002 Jan;66(1):112-9
references.pmid: 11751272
references.citation: Biol Reprod. 1997 Nov;57(5):1123-7
references.pmid: 9369179
references.citation: Nat Biotechnol. 2013 Aug;31(8):681-3
references.pmid: 23929336
references.citation: Mo Med. 2013 May-Jun;110(3):212-5
references.pmid: 23829105
references.citation: Nat Biotechnol. 2013 Mar;31(3):227-9
references.pmid: 23360964
references.citation: J Gen Virol. 2010 Jul;91(Pt 7):1659-67
references.pmid: 20410315
references.citation: Cloning Stem Cells. 2003;5(4):233-41
references.pmid: 14733743
references.citation: Mol Reprod Dev. 2010 Dec;77(12):1016-30
references.pmid: 20925087
references.citation: Cell. 2013 May 9;153(4):910-8
references.pmid: 23643243
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12013-7
references.pmid: 21730124
references.citation: Nature. 2012 Feb 15;482(7385):331-8
references.pmid: 22337052
references.citation: Transgenic Res. 2010 Aug;19(4):611-20
references.pmid: 19937273
references.citation: Proc Natl Acad Sci U S A. 2014 May 20;111(20):7260-5
references.pmid: 24799706
references.citation: Nature. 1985 Jun 20-26;315(6021):680-3
references.pmid: 3892305
references.citation: Biol Reprod. 2009 Sep;81(3):525-30
references.pmid: 19386991
references.citation: J Am Vet Med Assoc. 2005 Aug 1;227(3):385-92
references.pmid: 16121604
references.citation: Mol Reprod Dev. 2009 May;76(5):490-500
references.pmid: 19090011
references.citation: Science. 2002 Feb 8;295(5557):1089-92
references.pmid: 11778012
references.citation: Mol Reprod Dev. 2011 Oct-Nov;78(10-11):879-91
references.pmid: 21671302
references.citation: Cell Reprogram. 2011 Jun;13(3):191-204
references.pmid: 21548827
references.citation: Reprod Biol Endocrinol. 2003 Nov 07;1:82
references.pmid: 14613542
references.citation: Cloning Stem Cells. 2002;4(2):131-45
references.pmid: 12171705
references.citation: Cell. 2014 Feb 13;156(4):836-43
references.pmid: 24486104
references.citation: Adv Nutr. 2011 May;2(3):293-4
references.pmid: 22332062
references.citation: Proc Natl Acad Sci U S A. 1985 Jul;82(13):4438-42
references.pmid: 3892534
references.citation: Nucleic Acids Res. 1992 Jun 11;20(11):2902
references.pmid: 1614888
references.citation: J Virol. 2013 Sep;87(17):9538-46
references.pmid: 23785195
references.citation: Cell Res. 2014 Mar;24(3):372-5
references.pmid: 24481528
references.citation: Science. 2013 Feb 15;339(6121):819-23
references.pmid: 23287718
references.citation: Nat Biotechnol. 2002 Mar;20(3):251-5
references.pmid: 11875425