靶向猪基因组单链向导RNA快速筛选以及利用图案微阵列收获单克隆细胞的研究

Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal

规律性短重复回文序列簇(CRISPR)和CRISPR辅助蛋白9(Cas9)构成的CRISPR/Cas9基因编辑技术快速推进了基因修饰猪作为医学研究模式动物的广泛应用。而高效的靶基因单链向导RNA(sgRNA)是利用CRISPR/Cas9技术进行基因编辑成功的关键,对于猪等繁殖周期较长的大动物,则需要在实施动物实验前,在体外筛选出高效的sgRNA以避免时间和资源成本浪费。另外,如何高效获得阳性基因编辑单克隆细胞是目前尚待解决的难题。本研究建立了靶向猪基因组的sgRNA快速筛选方法,利用荧光载体富集基因编辑细胞,同时探索利用图案微阵列培养技术快速获得单克隆细胞的方法,在此基础上高效获得延胡索酰乙酰乙酸酶(Fah)基因编辑细胞,为后续生产作为人类肝细胞生物反应器的Fah基因敲除猪奠定基础。

The emergence of regular short repetitive palindromic sequence clusters(CRISPR) and CRISPRassociated 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 in vitro 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 Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.