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

ABSTRACT

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 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.