Gao Mengyu, Zhu Xinglong, Wang Shisheng, Zhang Bingqi, Zhang Yunlin, He Yuting, Zhou Yanyan, Li Shun, Yang Guang, Liao Guangneng, Bao Ji, Bu Hong
Department of Pathology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;Institute of Clinical Pathology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China.
Institute of Clinical Pathology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC; West China Hospital, Sichuan University, Chengdu 610041, P.R.China.
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2021 Feb 25;38(1):111-121. doi: 10.7507/1001-5515.202006032.
The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained gene edited cells, laying a foundation for the subsequent production of knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.
规律成簇短回文重复序列（CRISPR）和CRISPR相关蛋白9（Cas9）基因编辑技术的出现极大地推动了转基因猪的广泛应用。高效的单向导RNA（sgRNA）是使用CRISPR/Cas9技术进行基因编辑成功的关键。对于像猪这样繁殖周期长的大型动物，在进行动物实验前筛选出高效的sgRNA以避免浪费时间和资源成本是很有必要的。此外，如何高效获得阳性基因编辑单克隆细胞是一个有待解决的难题。在本研究中，建立了一种针对猪基因组的快速sgRNA筛选方法，并且我们快速获得了基因编辑细胞，为后续生产作为人肝细胞生物反应器的基因敲除猪奠定了基础。同时，探索了使用图案微阵列培养技术获得单克隆细胞的方法。
