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Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos

Biology of reproduction, 2014-09, Vol.91 (3), p.78 [Peer Reviewed Journal]

2014 by the Society for the Study of Reproduction, Inc. ;ISSN: 0006-3363 ;EISSN: 1529-7268 ;DOI: 10.1095/biolreprod.114.121723 ;PMID: 25100712

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Title:
Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos
Author: Whitworth, Kristin M ; Lee, Kiho ; Benne, Joshua A ; Beaton, Benjamin P ; Spate, Lee D ; Murphy, Stephanie L ; Samuel, Melissa S ; Mao, Jiude ; O'Gorman, Chad ; Walters, Eric M ; Murphy, Clifton N ; Driver, John ; Mileham, Alan ; McLaren, David ; Wells, Kevin D ; Prather, Randall S
Subjects: Animals ; Animals, Genetically Modified - genetics ; Animals, Genetically Modified - physiology ; Antigens, CD - genetics ; Antigens, CD - metabolism ; Antigens, CD1d - chemistry ; Antigens, CD1d - genetics ; Antigens, CD1d - metabolism ; Antigens, Differentiation, Myelomonocytic - genetics ; Antigens, Differentiation, Myelomonocytic - metabolism ; Blastocyst - physiology ; Cell Line ; CRISPR-Cas Systems ; Embryo Culture Techniques - veterinary ; Embryo Transfer - veterinary ; Embryo, Mammalian - physiology ; Female ; Fertilization in Vitro - veterinary ; Gene Deletion ; Genetic Engineering - adverse effects ; Genetic Engineering - methods ; Genetic Engineering - veterinary ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; In Vitro Oocyte Maturation Techniques - veterinary ; Male ; Mutation ; Nuclear Transfer Techniques - veterinary ; Oocytes - physiology ; Receptors, Cell Surface - antagonists & inhibitors ; Receptors, Cell Surface - genetics ; Receptors, Cell Surface - metabolism ; Sus scrofa - genetics ; Sus scrofa - physiology ; Transgenes
Is Part Of: Biology of reproduction, 2014-09, Vol.91 (3), p.78
Description: 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.
Publisher: United States
Language: English
Identifier: ISSN: 0006-3363
EISSN: 1529-7268
DOI: 10.1095/biolreprod.114.121723
PMID: 25100712
Source: MEDLINE
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Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos

2014 by the Society for the Study of Reproduction, Inc. ;ISSN: 0006-3363 ;EISSN: 1529-7268 ;DOI: 10.1095/biolreprod.114.121723 ;PMID: 25100712

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