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| Cloning of Upstream Regulatory Region of Brucella Resistance GeneIRF3 and Screening of Gene Edited Cell Colonies of Sheep (Ovis aries) |
| WANG Yuan-Yuan1, YE Li-Jun1, ZHU Yan-Yan1, CHEN Yu-Xin1, SHANG Fei-Fei1, LI Ting-Ting2, WANG Hai-Tao2,*, LIU Qiu-Yue2,* |
1 School of Life Science, Bengbu Medical College, Bengbu 233030, Anhui, China;
2 Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China |
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Abstract Brucellosis is one of the most serious infectious diseases in sheep (Ovis aries) and goats (Capra hircus) husbandry. In goats, mutations located at upstream regulatory region of interferon regulatory factor 3 (IRF3) gene have benefit for their resistance to brucellosis infection. IRF3 gene is highly homologous between sheep and goats, and same mutations of IRF3 can be obtained by gene editing in sheep. Aiming to obtain IRF3 edited cell colonies and supply donor cells to generate cloned Brucella resistant sheep by nuclear tranfer. The upstream open reading frame (uORF) region of sheep IRF3 gene was modified by gene editing mediated by CRISPR/Cas9 and prime editing (PE) technology. According to the reference sequence of sheep IRF3 in NCBI database, primers were designed, and the DNA sequence of IRF3 gene in Hu sheep was cloned; Small guide RNA (sgRNA) targeting IRF3-uORF and primer editing sgRNA (pegRNA) sequence were designed based on cloned sequences. All sgRNAs were constructed into the expression vector. Fetal fibroblast cell lines derived from Hu sheep were transfected by CRISPR/Cas9 or PE expression vectors. After monoclonal cell screening, two positive monoclonal cell lines with 10 bp deletion in IRF3-uORF region were successfully obtained by CRISPR/Cas9 but no positive colonies were obtained by PE. This study provide new ideas for combining somatic cloning to obtain new Brucella-resistant sheep germplasm at animal level.
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Received: 26 May 2022
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Corresponding Authors:
* wanght@genetics.ac.cn; qyliu@genetics.ac.cn
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