Fig. 4: The main principles of CRISPR/Cas9 genome editing.

CRISPR-associated nucleases (Cas proteins) are recruited to generate target-specific double-strand breaks (DSBs) through specific sequence recognition of the single guide RNA (sgRNA) with the target sequence of the genome and the presence of a protospacer-adjacent motif [PAM] sequence. DSBs are mainly repaired by endogenous repair pathways, including nonhomologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ facilitates joining two DNA fragments without the need for exogenous homologous DNA, resulting in small random insertions or deletions at the cleavage site. Consequently, the expression of the target gene is disrupted due to the production of frameshift mutations or premature stop codons during NHEJ-based repair. On the other hand, HDR facilitates precise gene insertion or replacement through the presence of donor DNA that contains a desired sequence and sequence homology at the DSB site. Created with BioRender.com (Agreement number : MB26ML0B76).