Fig. 2

The molecular mechanism of CRISPR-Cas9: SgRNA consists of tracrRNA and crRNA. The crRNA contains a 20-base recognition sequence and an additional sequence that complements tracrRNA. TracrRNA is hybridized with crRNA and combined with the Cas9 protein to form the CRISPR-Cas9/sgRNA complex, which produces DSBs at the target sites of the genome. SgRNA is often designed to contain two key fragments: a double-stranded RNA structure that binds Cas9 at the 3'-end and a guide sequence that binds the target DNA sequence at the 5'-end. SgRNA can recognize a specific sequence in the genome. The Cas9 protein is an endonuclease containing two domains (RuvC and HNH). The RuvC domain cleaves the noncomplementary DNA strand, while the HNH domain cleaves the complementary DNA strand. After DSBs are formed, either the NHEJ pathway or HDR pathway is activated. The NHEJ pathway often leads to insertions/deletions (Indel) and an open reading frame shift of the target gene. In contrast, the HDR pathway requires a donor DNA template to repair DSBs. The donor DNA template is used to insert the correct DNA sequence precisely into the target site