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Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer
- Source :
- Cellular and Molecular Life Sciences. 72:1175-1184
- Publication Year :
- 2014
- Publisher :
- Springer Science and Business Media LLC, 2014.
-
Abstract
- The domestic pig has been widely used as an important large animal model. Precise and efficient genetic modification in pig provides a great promise in biomedical research. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been successfully used to produce many gene-targeted animals. However, these animals have been generated by co-injection of Cas9 mRNA and single-guide RNA (sgRNA) into one-cell stage embryos, which mostly resulted in mosaicism of the modification. One or two rounds of further breeding should be performed to obtain homozygotes with identical genotype and phenotype. To address this issue, gene-targeted somatic cells can be used as donor for somatic cell nuclear transfer (SCNT) to produce gene-targeted animals with single and identical mutations. In this study, we applied Cas9/sgRNAs to effectively direct gene editing in porcine fetal fibroblasts and then mutant cell colonies were used as donor to generate homozygous gene-targeted pigs through single round of SCNT. As a result, we successfully obtained 15 tyrosinase (TYR) biallelic mutant pigs and 20 PARK2 and PINK1 double-gene knockout (KO) pigs. They were all homozygous and no off-target mutagenesis was detected by comprehensive analysis. TYR (-/-) pigs showed typical albinism and the expression of parkin and PINK1 were depleted in PARK2 (-/-)/PINK1 (-/-) pigs. The results demonstrated that single- or double-gene targeted pigs can be effectively achieved by using the CRISPR/Cas9 system combined with SCNT without mosaic mutation and detectable off-target effects. This gene-editing system provides an efficient, rapid, and less costly manner to generate genetically modified pigs or other large animals.
- Subjects :
- Swine
Somatic cell
CRISPR-Associated Proteins
Molecular Sequence Data
Mutant
Mutagenesis (molecular biology technique)
Biology
medicine.disease_cause
Gene Knockout Techniques
Cellular and Molecular Neuroscience
medicine
Animals
CRISPR
Molecular Biology
Cells, Cultured
Pharmacology
Mutation
Base Sequence
Cas9
Cell Biology
Fibroblasts
Molecular biology
Domestic pig
Phenotype
Gene Targeting
Molecular Medicine
Somatic cell nuclear transfer
CRISPR-Cas Systems
Genetic Engineering
RNA, Guide, Kinetoplastida
Subjects
Details
- ISSN :
- 14209071 and 1420682X
- Volume :
- 72
- Database :
- OpenAIRE
- Journal :
- Cellular and Molecular Life Sciences
- Accession number :
- edsair.doi.dedup.....1f31fd38e150024add0edb29685704f1