The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.
RNA介导的CRISPR/Cas9基因编辑系统由单链引导RNA(sgRNA)与核酸酶Cas9构成。在细胞内，sgRNA能够按照碱基互补配对的原则引导Cas9与靶点结合，由Cas9切割目标DNA，造成双链DNA断裂(double stranded break, DSB)。在随后的DNA修复过程中，细胞主要进行非同源末端连接(non-homologous end joining, NHEJ)或在有修复模板存在的情况下进行重组修复(homology directed repair, HDR)。如果将CRISPR/Cas9系统以及修复模板通过显微注射的方式导入大鼠的胚胎内，就能借助细胞的修复机制实现大鼠胚胎的基因编辑，由此构建各种基因修饰大鼠模型。本文详细介绍了利用CRISPR/Cas9基因编辑技术构建大鼠模型的具体操作步骤，以期为相关领域的科研人员提供一种大鼠基因修饰模型的构建方法。.
Keywords: CRISPR/Cas9; gene knockout; genome editing; rat.