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Genomics


accurate and stable engineering of human genomes without the introduction of potentially confounding or dangerous off-target errors or mutations; and improving the speed of gene-edit- ing still comes at a cost of precision at this time. Modern gene-editing technologies currently fall into two broad categories, those that rely exclu- sively on homologous recombination, a natural DNA-repair mechanism, to perform endogenous DNA alterations; and those that stimulate locus specific events as a consequence of introducing double strand DNA breaks. Each approach has its advantages: the latter allows the rapid and efficient ‘knock-out’ of specific genes, but also introduces unwanted off-target cuts in the genome and is com- paratively inefficient at performing subtle ‘knock- ins’ of disease causing point mutations; and the for- mer, which is currently less efficient at performing gene knock-outs, but does not introduce any off- target events and routinely can perform any genom- ic alteration, large or small at any target locus. The principle techniques are discussed further:


Linear double-stranded DNA homology vectors: This technique relies solely on homologous recom- bination (HR) and has been around for more than 10 years to create precision transgenic ‘knock-in’ and ‘knock-out’ mice. Vectors are simple stretches of homology to the target locus with a selectable marker in the middle (Figure 1). While this approach is inefficient compared to other more recent techniques, because mouse ES cells have very high natural rates of HR, this technique is perfectly adequate and still used commonly today. However, in human or other mammalian somatic cell-types, which have a far lower rate of homologous recom- bination, this technique is too unwieldy and has been abandoned in favour of more contemporary approaches that stimulate HR in some way.


Zinc-finger nucleases: These are relatively bespoke hybrid vectors that combine an adaptable, sequence specific Zinc-finger DNA-recognition domain, fused to a dimerisation-dependent nucle- ase, usually Fok1. When two zinc-finger nucleases (ZFNs) co-locate at a bipartite recognition sequence they create a dsDNA break, typically in both alleles, and thus elicit the rapid, permanent and relatively specific (compared to siRNA) dele- tion of a target gene. Absolute specificity is hard to achieve, and typically many off-target dsDNA breaks are introduced as well as the intended one. For human disease modelling and the creation of highly characterised bioproducer cell-lines, where complete precision is required, this is a significant


Drug Discovery World Spring 2011


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