Eukaryotic cells repair DNA double-stranded breaks (DSBs) through two primary pathways: end-joining or resection-mediated repair (Fig. 1) [1], [2], [3]. Non-homologous end-joining (NHEJ) serves as a major end-joining pathway, initiated by the heterodimeric Ku70/80 complex. Ku70/80 binds to free DNA ends and recruits DNA-PKcs to the damaged site, forming a DNA-PK holoenzyme. Subsequently DNA ligase IV and XRCC4 are recruited to ligate broken ends. Depending on the nature of the DNA ends, additional end-processing factors, such as nuclease ARTEMIS and polymerases μ and λ, might be required. Since NHEJ does not require a homologous template, it is active during the entire interphase, except middle-to-late S-phase, when homologous recombination (HR) becomes a predominant pathway to repair DSBs.

HR, the major resection-mediated repair pathway, is initiated by the CtIP-stimulated endonuclease activity of MRE11, which further generates 3’-overhang single-stranded DNA (ssDNA) through its 3’ to 5’ exonuclease activity (Fig. 1). This process known as a short-range resection can be further extended to long-range resection by EXO1 or DNA2/BLM. Initially, resected DNA ends are coated by the oligomeric RPA complex, which is subsequently replaced by RAD51 recombinase. RAD51-coated 3’ssDNA then invades the duplicated sister chromatid DNA, utilizing it as a template to restore the original DNA sequence. Tumor suppressors, BRCA1 and BRCA2 facilitate end-resection and RAD51 coating, respectively. Due to the requirement of a homologous template, HR is restricted to the S and G2 phases of the cell cycle, when a sister chromatid is available.

Of note, NHEJ can also depend on resection, which is mechanistically distinct from the resection used in HR [4], [5]. Resection-dependent NHEJ has slower repair kinetics than resection-independent NHEJ and repair damage in heterochromatic DNA or complex DSBs. The resection is initiated by Plk3-mediated phosphorylation of CtIP, enhancing its interaction with BRCA1, and requires nucleases MRE11, EXO1, and ARTEMIS.

Alternatively, resected DNA can undergo polymerase-θ-mediated end-joining (TMEJ) or single-strand annealing (SSA). In cases where the resected DNA has a microhomology sequence (2-8 nucleotides) with the other half of the broken DNA, polymerase-θ (Polθ) can anneal these sequences and extend the 3’-end of annealed ssDNA. For its use of microhomologies, it is sometimes called microhomology-mediated end-joining (MMEJ). However, it is important to mention that not all MMEJ depends on Polθ⊡

Of note, although NHEJ does not require any homology at the breakpoint, it can utilize 1-4 nucleotide microhomologies to repair the break [6], [7]. The transient base-stacking generated by annealing at microhomologies might assist in the synapsis of two DNA ends during NHEJ [2], [8]. Finally, when there is more extensive homology between broken halves of the DNA RAD52-mediated SSA can be activated [9]. In both TMEJ and SSA, the intervening sequence between homologous sequences is lost, resulting in deletions.

DNA DSB repair plays a crucial role in genome editing applications using CRISPR-Cas9. Originally discovered as a bacterial adaptive immune system, CRISPR-Cas9 has become a widely utilized tool for genome-engineering [10]. Cas9, a site-specific endonuclease, relies on the gRNA sequence for its specificity. Two separate domains of Cas9 are responsible for the cleavage of each DNA strand: the HNH domain cleaves the target strand, while the RuvC domain cleaves the non-target strand. Mutation in either of these domains (D10A or H840A/N863A) produces a Cas9 nickase (nCas9) capable of inducing site-specific single-stranded breaks. Alternatively, mutation in both domains yields a nuclease-dead Cas9 (dCas9), which retains target-binding activity. Cas9 requires a protospacer-adjacent motif (PAM), a short stretch of DNA, to bind the target sequence. For widely used Cas9 from Streptococcus pyogenes (SpCas9), PAM is 5’-NGG adjacent to the 3’-end of the non-target DNA strand.

Apart from genome engineering, Cas9 serves as a valuable tool for understanding DNA DSB repair mechanisms [11] and has several advantages over conventional methods used to generate DSBs, such as ionizing radiation, chemical mutagens, and restriction enzymes [11], [12]. For example, IR generates single strand breaks (SSBs), oxidized bases, and abasic sites in addition to DSBs, making it difficult to use to study DSB-specific responses. In addition, both IR and chemical mutagens induce DSB throughout the genome, making it challenging to spatially control DSB induction. On the other hand, restriction enzymes, such as AsisI, generate DSBs at defined locations in the genome, however, have limited programmability. In contrast, Cas9-induced DSB can be controlled both spatially and temporally [13].

Cas9 generates predominantly blunt-ended DSBs and less frequently breaks with 1 nucleotide 5’-overhangs [14]. Consistent with the notion that NHEJ is the most active repair pathway, Cas9-induced breaks are mainly repaired by NHEJ [14], [15], [16]. However, multiple repair pathways can repair the Cas9-induced break on a single locus, albeit with different kinetics [17]. Interestingly, mutagenic repair of Cas9-induced breaks was comparable in WT and LIG4 KO cells [18]. Here, Pol-Q was responsible for the mutagenic repair of the majority of breaks in the absence of LIG4 [18].

Nonetheless, Cas9-mediated cleavage cannot perfectly recapitulate physiological DSBs. First, the repair of Cas9-induced breaks appears to be relatively slow and mutagenic [17]. Next, Cas9 repeatedly cleaves the DNA until PAM or PAM-proximal sequence is disrupted, indicating, that the mainly observed repair outcome will be mutagenic NHEJ or TMEJ. In addition, to make a DSB, Cas9 unwinds DNA and generates an RNA-DNA hybrid R-loop structure. Importantly, the R-loop made by dCas9 is shown to be mutagenic in yeast [19]. Thus Cas9-based studies should be conducted in combination with the above-mentioned DSB-inducing strategies. Nonetheless, Cas9 provided valuable insights into DNA DSB repair as will be highlighted in this review.