Rosa26 Locus Supports Tissue-Specific Promoter Driving Transgene Expression Specifically in Pig

Kong, Qingran; Hai, Tang; Ma, Jing; Huang, Tianqing; Jiang, Dandan; Xie, Bingteng; Mei-ling, WU; Wang, Jiaqiang; Song, Yuran; Wang, Ying; He, Yilong; Sun, Jialu; Hu, Kui; Guo, Runfa; Wang, Liu; Zhou, Qi; Mu, Yanshuang; Z, Liu

doi:10.1371/journal.pone.0107945

Cited by 27 publications

(26 citation statements)

References 34 publications

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“…The sequence of the pig ROSA26 (pROSA26) locus has been completely characterized, and the pRosa26 promoter has been identified 8 . This porcine endogenous promoter is suitable for driving exogenous gene expression in a high and stable manner by avoiding DNA methylation.…”

Section: Introductionmentioning

confidence: 99%

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Xie

Pang

Wang

et al. 2017

Sci Rep

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Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.

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Section: Introductionmentioning

confidence: 99%

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Xie

Pang

Wang

et al. 2017

Sci Rep

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“…Historically, genetically modified pigs were generated either via random integration 5 or conventional homologous recombination. 6 However, random insertions often cause variable expression levels of the transgene, 7 whereas conventional HR targeting is extremely inefficient, 8 and the insertion of antibiotic selection cassettes has raised concern about their clinical prospects.…”

Section: Introductionmentioning

confidence: 99%

Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

Wang

Tang

Liu

et al. 2016

Molecular Therapy - Nucleic Acids

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Precise genome editing in livestock is of great value for the fundamental investigation of disease modeling. However, genetically modified pigs carrying subtle point mutations were still seldom reported despite the rapid development of programmable endonucleases. Here, we attempt to investigate single-stranded oligonucleotides (ssODN) mediated knockin by introducing two orthologous pathogenic mutations, p.E693G for Alzheimer's disease and p.G2019S for Parkinson's disease, into porcine APP and LRRK2 loci, respectively. Desirable homology-directed repair (HDR) efficiency was achieved in porcine fetal fibroblasts (PFFs) by optimizing the dosage and length of ssODN templates. Interestingly, incomplete HDR alleles harboring partial point mutations were observed in single-cell colonies, which indicate the complex mechanism of ssODN-mediated HDR. The effect of mutation-to-cut distance on incorporation rate was further analyzed by deep sequencing. We demonstrated that a mutation-to-cut distance of 11 bp resulted in a remarkable difference in HDR efficiency between two point mutations. Finally, we successfully obtained one cloned piglet harboring the orthologous p.C313Y mutation at the MSTN locus via somatic cell nuclear transfer (SCNT). Our proof-of-concept study demonstrated efficient ssODN-mediated incorporation of pathogenic point mutations in porcine somatic cells, thus facilitating further development of disease modeling and genetic breeding in pigs.

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“…The most preferred integration site used for transgene insertionis the Rosa26 locus in mice. As in mice, the human and rat Rosa26 loci were identified and successfully targeted using traditional HR 13 . There have reported that knock-in of a long DNA fragment via homology-independent DNA repair can be achieved in zebrafish using the CRISPR/Cas9 system.…”

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confidence: 99%

Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system

Wei

Lian

et al. 2016

Sci Rep

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Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.

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Rosa26 Locus Supports Tissue-Specific Promoter Driving Transgene Expression Specifically in Pig

Cited by 27 publications

References 34 publications

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system

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