In a classic switcheroo, a talented research team led by ChongHua Ren inserted a repeat-flanked Zinc Finger Nuclease (ZFN) binding site right in the middle of the transcription factor Gal4, shifting control of Gal4 expression to capricious homologous recombination events.
ZFNs edit the genome at specific locations by cutting DNA at the endonuclease site. When that site is surrounded by repeats, the cut can cause single-stranded annealing and homologous recombination. Although researchers knew that this event is dependent upon the length of the repeats, no one had figured out the minimum length required for a ZFN-mediated recombination event.
To address this knowledge gap, the researchers devised a simple and efficient yeast-based ZFN screening system. Specifically, they designed reporter plasmids containing direct repeats of varying lengths (10-164bp) flanking the ZFN binding site, where recombination could either occur spontaneously (tricking researchers into a false positive ZFN cutting event) or could be mediated by a true ZFN-induced DNA cut.
Using this system, they performed a systematic check to discover the minimum repeat length necessary for a spontaneous or ZFN-induced homologous recombination event to occur.
The advancements in ZFN biochemistry made by the group are multi-fold
- First, they developed a novel, optimized ZFN screening system that outcompetes the current LacZ reporter technology
- Second, they discovered that the minimum direct repeat length necessary to induce spontaneous or ZFN-mediated homologous recombination is 20bp,
- Lastly, they found that a 30bp repeat achieves more efficient ZFN-induced recombination and almost completely eliminates spontaneous recombination that results in false positives during ZFN screening.
Cut to the details at Mol. Biol. Rep., 2014