Indel profiling of primary hepatocytes by deep sequencing

CRISPR/Cas9 has proven to be an efficient tool for gene editing both in vitro and in vivo. The aim of this study is to evaluate editing efficiency of various genes targted in Rosa26-Cas9 knock-in mice. Overall design: Efficiency of in vivo genome editing was assessed using for hepatocytes isolated from the livers of Cas9-EGFP mice to which adeno-assocaited viruses (AAVs) packaged with sgRNAs targeting various genes were injected. Genemic regions containing each cut site were amplified by PCR, and libraries were prepared for deep sequencing.CRISPR/Cas9已被证实为一种可用于体外（in vitro）和体内（in vivo）基因编辑的高效工具。本研究旨在评估Rosa26-Cas9敲入小鼠中各靶向基因的编辑效率。整体实验设计：向Cas9-EGFP小鼠注射包装有靶向不同基因的单向导RNA（single guide RNA，sgRNA）的腺相关病毒（adeno-associated virus，AAV），随后分离其肝脏来源的肝细胞，以此评估体内基因组编辑效率。通过聚合酶链式反应（Polymerase Chain Reaction，PCR）扩增包含各切割位点的基因组区域，并构建文库用于深度测序。