However, if the DNA break is introduced in the presence of a targeting vector, then this altered DNA sequence is utilized as the repair template, with the result that the exogenous change is now introduced into the DNA. Thus, the combination of a site-specific nuclease and a targeting vector will dramatically increase the efficiency with which the desired change is introduced (Figure 1).
Although a site-specific nuclease may increase the frequency of HR at a desired site by several orders of magnitude, in many cases efficiencies are still not extremely high and one must have some way to select for the desired mutation. Typically, this is done by using a selectable marker—that is, a gene encoding a protein that renders the cell identifiable (e.g., it is now resistant to a drug or is fluorescent, such that drug treatment or cell sorting can be used to kill undesired non-altered cells or select and concentrate the desired cells).
This strategy works quite well, but also introduces another complication as the presence of the selectable marker may interfere with the expression of the altered target gene. Thus, an ideal gene-editing system must include some mechanism by which this marker can be excised cleanly from the genome once the desired mutant cells have been selected.
Until recently, the typical solution has been to surround the selectable marker with the target sequences for the Cre recombinase. This enzyme, derived from a bacteriophage, catalyzes recombination between these target sites (known as loxP sites). If the loxP sites are in the same orientation, the intervening selectable marker gene is excised. However, this process is not clean as it leaves behind one of the 34 base pair loxP sites. This remaining “footprint” is not ideal when creating a mutation, especially for human gene therapy or regenerative medicine applications.
Fortunately, a novel solution now exists, utilizing the piggyBac™ DNA transposon.
DNA transposons, sometimes called “jumping genes”, are naturally occurring mobile DNA elements that encode the gene for an enzyme, a transposase, that is flanked by a pair of inverted terminal repeats (ITRs). The transposase recognizes these ITRs and catalyzes the excision of the transposon (and anything encoded between its ITRs) from its resident site and occasionally then inserts it elsewhere within the genome.
While most transposons leave behind some mutation when they are excised, the piggyBac transposon is unique in that its excision restores the sequence of the DNA that existed before the insertion occurred. That is, excision is footprint-free. The transposase gene does not need to be encoded between the ITRs, but rather can be provided in trans and catalyze the footprint-free insertion and excision of any DNA sequence flanked by piggyBac ITRs.
Thus, by flanking selectable markers with ITRs and placing this artificial transposon in a targeting vector, one can then both select for the desired gene modification and then completely remove the selectable marker in a clean and footprint-free manner.
The stage is thus now set for rapid, efficient, and footprint-free gene alterations by combining a site-specific double-stranded nuclease with piggyBac-based selectable markers incorporated into a targeting vector.