The CRISPR gene editing revolution is less than a decade old, and scientists are still learning how best to deploy this ground-breaking technique. A team from the University of Illinois at Chicago has recently discovered why the technique sometimes fails, uncovering a novel insight into the new process that will hopefully make future research more efficient and effective. The CRISPR gene editing process is known to fail about 15 per cent of the time. These random failures have until now been a mystery to scientists who consistently work with the technique. When the technique is working properly, an enzyme called Cas9 is what actively makes the cuts to the desired position in a DNA strand. After the cut is made either a new desired sequence is added, or an unwanted section is simply removed, with the two cut points glueing themselves back together.
The new research revealed that when the CRISPR process fails it is because the Cas9 enzyme effectively sticks to the DNA cut point, blocking the subsequent DNA repair process. “We found that at sites where Cas9 was a ‘dud’ it stayed bound to the DNA strand and prevented the cell from initiating the repair process,” explains Bradley Merril, senior author on the new study. The study also found that when RNA polymerases collide with Cas9, they can force the enzyme to dislodge. This means that consistent strand selection can significantly improve the genome-editing technique’s efficiency. “I was shocked that simply choosing one DNA strand over the other had such a powerful effect on genome editing,” says Ryan Clarke, lead author of the study. “Uncovering the mechanism behind this phenomenon helps us better understand how Cas9 interactions with the genome can cause some editing attempts to fail and that, when designing a genome editing experiment, we can use that understanding to our benefit.”
While this kind of micro-mechanism discovery may seem somewhat academic, it is research such as this that is slowly building up a new body of knowledge in a nascent field that is still mostly uncharted territory. Bradley Merril explains that this discovery could help speed up the overall time Cas9 interacts with the DNA strand, which should make the process more efficient. “If we can reduce the time that Cas9 interacts with the DNA strand, which we now know how to do with an RNA polymerase, we can use less of the enzyme and limit exposure,” says Merrill. “This means we have more potential to limit adverse effects or side effects, which is vital for future therapies that may impact human patients.”
The study was published in the journal Molecular Cell.
Credit: Rich Hardy for New Atlas, 12 July 2018.