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| CRISPR/Cas9-mediated Site Specific Integration of Foreign Genes in Pig (Sus scrofa) PSP Locus |
| JIN Wei, DAI Min-Min, LI De-Juan, FAN Bao-Liang* |
1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China;
2 Hebei Animal Husbandry and Veterinary Institute, Baoding 071000, China;
3 College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
4 College of Animal Science and Technology, China Agricultural University, Beijing 100193, China |
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Abstract As a novel bioreactor, the salivary gland bioreactor specifically expresses various foreign proteins by using the regulatory region of salivary secretory proteins. The commonly used salivary secretory protein is parotid secretory protein (PSP).The aim of this study is to establish a method for site-specific integration of foreign genes into PSP gene using the CRISPR/Cas9 system and analyze the off-target effect of the selected gRNA on PSP gene. Firstly, sgRNA sequences were designed online according to PSP gene sequence and 5 sgRNA sequences were selected. Then, using sgRNA in vitro transcription kit and Cas9 in vitro digestion kit, the sgRNA sequence with high in vitro activity which targeting PSP gene were screened out. At the same time the co-expression vectors of pCas9-sgRNA for sgRNA and Cas9 and a targeting vector pMD19-5'arm-NeoR-3'arm carrying the expression structure of neomycin resistance gene (NeoR) were constructed. Finally, sgRNA and Cas9 co-expression vector and target vector were co-transfected into porcine kidney cells (PK-15 cells), screened with neomycin, and single cell clones were isolated. Positive cell clones which was knocked-in neomycin resistance gene (NeoR) were identified by PCR and sequencing, the off-target effects of every sgRNA were analyzd. The results of in vitro enzyme digestion showed that sgRNA1, sgRNA3, sgRNA4 and sgRNA5 had higher activity in vitro. Sequencing results indicated that the 4 sgRNAs were successfully targeted the forigen gene into the PSP gene of Sus scrofa. The knock-in efficiency of sgRNA1 and sgRNA5 was 22.7% (5/22) and 26.1% (6/23), with 1 potential off-target site had off-target effect; The efficiency of sgRNA3 and sgRNA4 was 50.0% (12/24) and 42.1% (8/19), with no off-target effect at the 5 potential off-target sites. In this study, the method for gene editing of pig PSP gene was successfully established, and screening for sgRNAs that efficiently guide foreign genes into the pig PSP gene provides basic data for further research on transgenic Sus scrofa.
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Received: 03 February 2019
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Corresponding Authors:
fanbl119@vip.sina.com
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