What are the mechanisms of the CRISPR/Cas9 system?

The CRISPR/Cas-9 genome editing process involves three main steps: recognition, cleavage, and repair. Initially, The designed sgRNA identifies the target sequence within the gene of interest, specifically through its 5' crRNA complementary base pair component. Without the presence of sgRNA, the Cas-9 protein remains inactive. Upon locating the target site, Cas-9 creates double-stranded breaks (DSBs) at a site located three base pairs upstream of the protospacer adjacent motif (PAM) sequence. Once Cas-9 identifies the right PAM sequence, it initiates local DNA melting and forms an RNA-DNA hybrid. Following this step, Cas-9 becomes activated for DNA cleavage. The HNH domain of Cas-9 cleaves the complementary strand, while the RuvC domain cleaves the non-complementary strand, resulting in blunt-ended DSBs. In the last step, the cellular mechanisms of either non-homologous end joining or homology-directed repair mend the double-stranded break. Non-homologous end joining serves as the primary cellular repair mechanism for DSBs in DNA. It operates throughout all phases of the cell cycle, ensuring efficient repair of damaged DNA. This enzymatic process involves the direct ligation of broken DNA ends without the requirement for external DNA templates. Homology-directed repair is highly precise, particularly in the late S and G2 phases of the cell cycle. It requires a large quantity homologous DNA template to accurately restore damaged DNA sequences. These templates aid in accurately integrating genetic material at the specific site of the double-stranded break, enabling both insertion and replacement of genetic material.