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Establishment of CRISPR/Cas9-Mediated Knock-in System for Porcine Cells with High Efficiency

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Abstract

Since the birth of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, the new genome engineering technology has become a hot topic in the scientific community. However, for swine, the system of pig cells’ homology directed repair (HDR) is generally unstable and costly. Here, we aim to make knock-in of porcine cells more realizable. The Rosa26 locus was chosen for gene editing. Through the optimization of strategy, an efficient sgRNA was selected by TIDE analysis. Correspondingly, a vector system was constructed for gene insertion in pRosa26 locus by homologous recombination. A large percentage of cells whose gene is edited easily result in apoptosis. To improve the positive rate, culturing systems have been optimized. Sequence alignment and nuclear transfer confirmed that we got two knock-in cell lines and transgene primary porcine fetal fibroblasts (PFFs) successfully. Results showed that the gene editing platform we used can obtain genetically modified pig cells stably and efficiently. This system can contribute to pig gene research and production of transgenic pigs.

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Funding

This work was supported by grants from the Program of National Natural Science Foundation of China (31572399), The National Key Research and Development Program of China, Stem Cell and Translational Research (Grant No. 2016YFA0100203).

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Authors and Affiliations

  1. College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Juqing Zhang, Zhenshuo Zhu, Wei Yue, Jiaxin Li, Qiang Chen, Yuan Yan, Anmin Lei & Jinlian Hua

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  1. Juqing Zhang
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  2. Zhenshuo Zhu
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  3. Wei Yue
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  4. Jiaxin Li
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  5. Qiang Chen
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  6. Yuan Yan
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  7. Anmin Lei
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  8. Jinlian Hua
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Contributions

Juqing Zhang and Jinlian Hua designed the study and wrote the manuscript. Juqing Zhang performed most experiments. Zhenshuo Zhu, Wei Yue, Jiaxin Li, Qiang Chen, Yuan Yan, Anmin Lei analyzed the data.

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Correspondence to Jinlian Hua.

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Figure S1.

We sequenced the left side and found that the result was as expected. (A) PCR results showed that we got PFFs which occurred knockin. Primer KI-GFP-Left-F is on the genome upstream the left homologous arm, and primer KI-GFP-Left-R is targeting the insertion sequence. The length of the product is 2145 bp. (B) Sequence alignment results show that editing of purified cells is accurate. (PNG 2086 kb)

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Zhang, J., Zhu, Z., Yue, W. et al. Establishment of CRISPR/Cas9-Mediated Knock-in System for Porcine Cells with High Efficiency. Appl Biochem Biotechnol 189, 26–36 (2019). https://doi.org/10.1007/s12010-019-02984-5

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