**2. DSB repair pathways involved in genome editing**

The CRISPR/Cas9-mediated gene knock-out or knock-in is based on the mechanisms of DSB repair. To better understand the genome editing by the CRISPR/Cas9 method, we first provide an overview of the DSB repair mechanisms.-

DSBs are repaired by multiple mechanisms [30] (**Figure 1**). One of the major DSB repair mechanisms is nonhomologous end joining (NHEJ). NHEJ is the simplest method for DSB repair, MultiSite Gateway Technology Is Useful for Donor DNA Plasmid Construction in CRISPR/Cas9-Mediated… 85 http://dx.doi.org/10.5772/intechopen.80775

**Figure 1.** Multiple DSB repair pathways. The targeted DSB induced by CRISPR/Cas9 is repaired by NHEJ, MMEJ, SSA, or HR.-

in which the broken ends of the DNA are rejoined, and is a rapid and predominant DSB repair pathway in mammalian cells [31]. The DSB is accurately repaired to its normal state when the broken ends are protected during repair. However, if the broken ends are digested before rejoining, the DNA information at or around the DSB site is lost. Thus, NHEJ is an errorprone DSB repair pathway [32]. CRISPR/Cas9-mediated gene knock-out technology utilizes this mutagenic aspect of NHEJ, and thus requires only DSB induction in the targeted gene. Most of the DSBs are repaired accurately by NHEJ [33]. However, if the DSB site is repaired accurately, then the target DNA site seems to be attacked repeatedly by CRISPR/Cas9, until the site is broken and thus insensitive to the hybridization with the crRNA (**Figure 2**). This apparently enhances the knock-out efficiency by CRISPR/Cas9.-

The second major DSB repair pathway is homologous DNA recombination (HR) [34, 35] (**Figure 1**). In HR, DSBs are repaired by DNA strand exchange with the undamaged homologous DNA strand. In *E. coli* or yeast, HR is the predominant mechanism for DSB repair. In the first step of HR, the DSB ends are resected by a nuclease to generate 3′ single-strand (ss) DNA overhangs. Then, the ssDNA overhangs invade and anneal with the undamaged homologous DNA strand. New DNA is synthesized from the 3′ end of the invaded DNA as a primer, according to the sequence information of the undamaged DNA template, thereby

**Figure 2.** Gene knock-in or knock-out by DSB repair pathways. (A) Targeted gene disruption induced by NHEJ. (B) Targeted short DNA fragment insertion mediated by MMEJ or SSA. (C) Targeted long DNA fragment insertion mediated by HR.-

restoring the lost sequence information at the damaged sites. The branch point in the crossed DNA strands moves during the repair process. Finally, the crossed DNA strands are resolved by cutting and rejoining. Thus, the DNA information can be restored by HR repair even if the broken DNA ends are digested, such as by nucleases. Therefore, HR is a more precise DSB repair mechanism, as compared to NHEJ.-If the homologous DNA strand is available in the donor DNA, then the donor DNA is integrated into the damaged site by HR (**Figure 2**). Thus, HR is an important mechanism for a gene knock-in.-

DSBs are also repaired by other mechanisms, including microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) (**Figure 1**). Although MMEJ and SSA are mechanistically similar, they are distinct pathways with different repair proteins. Both MMEJ and-SSA use relatively short internal homologous sequences flanking both sides of the DNA break.-The length of the homologous sequence required for MMEJ is shorter than that for SSA.-Thefirst step of MMEJ or SSA is the resection of the DSB site, and thereby the homologous dsDNAregion becomes ssDNA.-In the next step, the ssDNA regions with the homologous sequenceare annealed, and the 3′ flaps of the nonhomologous region are removed. Finally, the gappedDNA regions are filled by DNA synthesis, and the resulting nicks are rejoined [36–39]. Both- repair pathways induce a DNA deletion at the damaged site, and therefore could contribute to the CRISPR-Cas9-mediated gene knock-out in the absence of a DNA donor [40, 41]. However,- when the donor DNA is provided, the repair pathways can be used for gene knock-in [42–44] (**Figure 2**).-
