DSBs commonly occur in eukaryotic cells due to a range of endogenous and exogenous factors. They are mainly repaired through HDR and NHEJ mechanisms which restore the duplex structure of DNA. For the HDR mechanism to operate, the organism needs to be diploid (even if diploidy is transiently achieved). This means that the HDR pathway requires a homologous reference sequence (with homology to sequences adjacent to the DNA lesion) to correct DSBs by incorporating the sequence available in the reference DNA strand. The homologous reference sequence can be engineered and exogenously supplied or can also come from the sister chromatid involved in meiosis I (when HDR is active during the late S and G2 phases of the cell cycle). In mammalian cells, the HDR mechanism involves the resection of each DSB strand in the 5’ to 3’ direction leading to the generation of 3′ overhangs with the subsequent strand foray of a homologous locus by the 3′ end. Homologous recombination is the most common form of HDR mechanism that requires the longest sequence homology for DNA repair. HDR pathway faithfully repairs without the formation of indels. Other HDR mechanisms include single-strand annealing (SSA) and breakage-induced replication.
However, in a haploid organism or in a diploid organism that is not in the S phase of the cell cycle, the homologous DNA strand might not be available for DNA repair. In such events, the NHEJ mechanism assists in DSB repair and assists in the survival of the organism. The NHEJ pathway has evolved to exhibit mechanistic flexibility, multi-functionality of enzymes, and diversity of substrates that can be joined by the end-to-end fusion. The first step of the NHEJ pathway involves the binding of a toroidal protein, Ku, at the two ends of the DNA at a DSB. This helps to dock the nuclease (resects DNA lesion), polymerase (adds nucleotides in a template-independent fashion), and ligase (restores the integrity of DNA strands). NHEJ usually repairs DSBs faithfully but at times inserts or deletes (known as indels) about 1 to 10 base pairs from the site of the cut. Indels can lead to frameshift mutations that can disrupt the expression of the associated protein.
In addition to endogenous signals that activate DNA damage repair pathways, CRISPR and other gene editing technologies can also trigger the body’s natural DNA repair mechanisms to set in. For e.g., the CRISPR technique can be used to introduce cuts at targeted sites generating DSBs. This activates natural DNA repair mechanisms to repair the DSBs. Both HDR and NHEJ mechanisms are have been reported to repair DNA DSBs introduced by CRISPR technology. Although NHEJ can lead to the formation of random indels during DNA repair, HDR can help to achieve precisely repaired genetic edits such as base substitutions and knock-ins.