The stimulatory nature of DSBs for gene targeting is related to how cells naturally repair DSBs. DSBs are naturally occurring events that have the potential to cause chromosomal rearrangements or cell death. But cells have redundant mechanisms to repair DSBs. The two primary mechanisms are by non-homologous end-joining (NHEJ) and HR ().
Framework for DSB repair in mammalian cells.
In NHEJ, the broken ends are simply ligated back together. If the DSB is ‘clean’ and the broken ends are compatible, repair of a DSB by NHEJ will usually be non-mutagenic. But occasionally, repair of a clean DSB by NHEJ will be mutagenic. If the DSB is complex—creating ends that are not compatible, then repair by NHEJ will necessarily be mutagenic. That is, the repaired DNA will have sequence differences, either the insertion or deletion of nucleotides, which were not present in the original segment.
The second major mechanism that cells repair a DSB is by HR. Conceptually repair of a DSB by HR is by a ‘copy and paste’ mechanism. Mechanistically, repair of a DSB proceeds in the following fashion and has been reviewed elsewhere (82
). Here we briefly describe the synthesis-dependent strand-annealing (SDSA) model of DSB repair by HR (). First, the ends of the DSB are resected to generate 3′ single-stranded tails. The generation of these tails requires the Mre11/Rad50/Nbs1 multi-protein complex but the exact nuclease that generates the 3′ tails remains to be identified. These 3′ tails are protected from nucleolytic degradation by coating with RPA. The next step in HR is strand invasion. In this step, Rad51 displaces RPA from the single-stranded DNA (in conjunction with Rad52 and BRCA2) and the Rad51/ssDNA nucleofilament invades an undamaged homologous segment of DNA. Then primed DNA synthesis occurs as the undamaged DNA serves as a template for DNA replication with the invading strand serving as the primer. After DNA synthesis the complex becomes unraveled and the DNA strands pair with their original partners. The newly synthesized DNA then serves as a template for DNA replication for its partner strand with the result that a complete undamaged double-strand piece of DNA is formed. The DNA used as the template is unperturbed and this is why the mechanism can be conceptualized as ‘copy and paste’ rather than ‘cut and paste.’
Figure 6 ZFN-induced homology-directed DSB repair in mammalian cells. Initially, gene ‘a’ is undamaged. The repair of the gene by HR occurs after the induction of a DSB. The DSB may arise spontaneously such as by damage from reactive oxygen species (more ...)
The fidelity of the repair by HR depends on its choice of homologous DNA to use as a repair template. Usually, the cell uses the sister-chromatid as a template. Since the sister-chromatid is identical to the damaged chromosomal DNA using it as a template will result in a perfect form of repair, no matter how complex the original DNA break was. Rarely, however, the cell will use an alternative piece of homologous DNA as the repair template—either the homolog chromosome or some other random piece of homologous DNA. If there are sequence differences between the homologous template and the damaged DNA, then those differences will be transferred to the damaged allele. For example, loss of heterozygosity can occur if the chromosomal homolog is used as a repair template. Finally, if the cell uses an introduced extra-chromosomal segment of DNA as the repair template, then gene targeting will result. Thus, gene targeting is the result of the cell using its own endogenous repair mechanism to repair a DSB.
Random integration versus gene targeting
In gene targeting, an exogenous piece of DNA is introduced into the cell. An important aspect to understanding gene targeting is to consider what happens to that piece of DNA. There are four general outcomes: (i) It can be degraded in which case the introduction will be of no consequence in the long-term. (ii) It can be maintained as an episomal fragment. Unless it contains a mammalian origin of replication, such as that occurs on some Epstein–Barr virus (EBV)-based vectors, however, the piece of DNA will eventually be lost as the cell divides. On the other hand, if the cell is not dividing, then the fragment of DNA can be maintained episomally for long periods of time. This seems to be a frequent occurrence, e.g. when hepatocytes are infected with adeno-associated virus. (iii) It can be used as a template for the repair of a DSB by HR and subsequently lost. If this occurs, then gene targeting occurs as sequence information from the introduced DNA fragment is transferred to a homologous segment of the host genome. (iv) It can integrate randomly into the genome. In random integration, it is not just information that is transferred to the host genome, but the actual DNA fragment is integrated into the host genome. But because it is integrating in a non-homologous fashion, the integration of the DNA is fundamentally a mutagenic event. Surprisingly little is known about the mechanism of random integration in mammalian cells. Interestingly, random integration can occur at sites of DSBs—it appears that the cell uses the piece of DNA to ‘patch’ a break. When this occurs, there is no surrounding sequence homology to suggest that the integration was through an HR mechanism. Certain types of random integration also appear to be dependent on NHEJ. One of the appeals of gene targeting is that it is a precise way to manipulate the genome. In thinking about how to utilize gene targeting most effectively, one must not only think about how to increase the frequency that the extra-chromosomal DNA is used as a repair template for the repair of a DSB by HR, but also about how to decrease the frequency of the extra-chromosomal DNA integrating randomly and thereby causing undesired mutations.