Although this is considered the gold standard, this method can be costly, time consuming and laborious, even for a limited number of clones. Typically, the targeted exon is PCR amplified and cloned into a vector for bacterial single colony sequencing. In order to identify the exact sequence of the resulting alleles in selected clones, most laboratories use Sanger sequencing. These three indels can arise from colony formation that started from two cells as opposed to one, or more likely as a result of residual nuclease activity in a daughter cell resulting in an additional indel as this phenomenon is observed even under strict single cell sorting conditions. Even in diploid cells, three different indels are often observed. Since the indels typically introduced by the repair mechanisms are largely random, they are unlikely to be identical. In a diploid cell, next generation nuclease-mediated gene-silencing commonly results in either one or two indels.
Alternatively, if an oligo-nucleotide with a high degree of homology surrounding the strand break or nicks is introduced, the endogenous homology directed repair mechanism can use the oligo as a repair template, thereby allowing precise gene insertions or modifications 6. Depending on the nature of the Cas9 protein, this results in a DNA double strand break or a nick, leading to nucleotide insertions or deletions (indels) due to errors in the cell’s endogenous DNA repair mechanisms. The gRNA variable domain can be modified to target virtually any gene of interest, thereby localizing the system to a specific region of the genome. Briefly, CRISPR is a bipartite system comprised of an endonuclease domain entailing the Cas9 protein and a guide RNA (gRNA), which binds Cas9. The CRISPR mode of action has been previously described in great detail. CRISPR allows the rapid generation of gene knockouts or knock-ins in in vitro and in in vivo models, and finds a wide range of applications beyond gene editing 3, 4, 5.
Whilst the next generation gene editing tools, zinc finger nucleases and TALENs have been widely available 1, 2, the advent of the CRISPR-Cas9 system (CRISPR) augmented the accessibility of precise gene editing, leading to its ubiquitous adoption.
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