Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer
- 5.2k Downloads
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.
KeywordsAlbinism Animal model Genome engineering Parkinson’s disease
Clustered regularly interspaced short palindromic repeats
Porcine fetal fibroblast
PTEN-induced putative kinase 1
Somatic cell nuclear transfer
This study was supported by the Natural Science Foundation of China (Nos. 31301103 and 31360532) to H.Y. and J.X., the National Basic Research Program of China (973 program; No. 2011CB944203) to L. L.
Conflict of interest
The authors have declared no conflict of interest.
- 7.Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156(4):836–843CrossRefPubMedGoogle Scholar
- 14.Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, Ali Z, Del Turco D, Bentivoglio AR, Healy DG, Albanese A, Nussbaum R, González-Maldonado R, Deller T, Salvi S, Cortelli P, Gilks WP, Latchman DS, Harvey RJ, Dallapiccola B, Auburger G, Wood NW (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 304(5674):1158–1160CrossRefPubMedGoogle Scholar
- 18.Yang D, Yang H, Li W, Zhao B, Ouyang Z, Liu Z, Zhao Y, Fan N, Song J, Tian J, Li F, Zhang J, Chang L, Pei D, Chen YE, Lai L (2011) Generation of PPAR gamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res 21(6):979–982CrossRefPubMedCentralPubMedGoogle Scholar