Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 25

The genomes of Higher Eukaryotes and in particular mammalian genomes are quite resilient to genetic manipulation. It was only in the 1986 that the first gene targeting experiment to generate a functional Knock‐Out of the HPRT gene was published by scientists in Capecchi´s group (Thomas et al. 1986). The same targeting strategy was further optimized by Janish´s group leading to the establishment of a gene targeting pipeline in mESC where potentially any gene can be modified to generate genetically engineered mice models. Despite several attempts to further optimize the method to reduce homology arms length, it became evident that this gene targeting strategy in mESC requires long stretches of homology to the target sites to obtain an optimal efficiency of recombination. High frequency of recombined cells allows the isolation of engineered clones. An increase in the homology arm length by using targeting engineered BACs could further boost this efficiency (Yang and Seed 2003). One characteristic of this canonical gene targeting strategy is that most of the targeting events result in mono‐allelic insertion. Consequentially, additional gene targeting steps or animal breeding strategies are required to obtain bi‐allelic targeting. As discussed above, Recombineering has recently become the method of choice to generate the complex targeting cassettes with long homology arms needed for mammalian genome manipulation.

This classical gene targeting approach works relatively well in mESC and in DT40, a chicken bursal lymphoblast cell line (Buerstedde and Takeda 1991). It is still not clear to this day the reason why this approach is difficult to implement in other cell types. However, in 2002, David Russel lab showed that it is possible to use Adeno Associated Virus (AAV) to promote gene targeting in mammalian cell lines (Hirata et al. 2002). Despite the importance of this finding, this system is still laborious and pretty inefficient in inducing bi‐allelic gene targeting. A promising approach to introduce small indels/mutations by single‐stranded oligonucleotide in mammalian cells has been described by Kmiec´s lab using chimeric oligonucleotides, but it has not been extensively applied for the generation of cellular model due to its low efficiency and due to lack of reproducibility when targeting different loci (Cole‐Strauss et al. 1996).

Experiments from Jasin´s group clearly demonstrated that a targeted DSB induced by I‐SceI enzyme can overcome the anti‐recombinogenic feature of mammalian genome by promoting homologous recombination. This experiment was based on previous observations in budding yeast on mating‐type switching (Strathern et al. 1982) and on the model of recombination dependent on DSB generation (Szostak et al. 1983). There are two important findings as result of the work from Jasin´s group. The first one is that DSBs are preferentially repaired by NHEJ in mammalian cells and although it is difficult to evaluate the amount of perfect repair by precise NHEJ, imprecise NHEJ, using additional enzymes to process the broken ends of the DSBs, can result in template‐independent gene disruption. The second important finding is that DSBs can promote bi‐allelic DNA recombination if the break occurs in both alleles.