The CRISPR gene editing technique has had a dramatic influence on biomedical research, and has even been applied to a few humans to treat disease. Since its invention, scientists have improved, altered, and refined CRISPR-based genome-editing tools to apply them to a variety of different kinds of cells. In the basic version of CRISPR, an enzyme known as Cas9 and a molecule called a guide RNA are used to make a targeted cut in DNA, which is then repaired by cellular machinery. This can lead to mutations in DNA that can be studied. A genetic repair template that has a desired sequence can also be used with CRISPR so that when the cell fixes the cut, the desired sequence is then introduced.
This process can be fairly straightforward. But complexities can arise when this method is applied to cells that do not divide, because the cellular repair machinery can work a bit differently in non-dividing cells. So researchers wanted to learn more about what happens when CRISPR is used in cells that do not divide or only divide rarely, such as many neurons.
Scientists applied the CRISPR technique, but did so with carefully measured quantities of DNA-editing reagents. The team, which included CRISPR pioneer and Nobel prize winner Jennifer Doudna, Ph.D. of the Gladstone Institutes, created nanoparticles to send these controlled levels of CRISPR reagents into neuronal cells that do not divide and induced pluripotent stem (iPS) cells that do divide.
This work showed that the same levels of Cas9 targeting the same sequence of DNA produced significantly different results in the different cell types. In neurons, the Cas9 editing went on for as long as one month, which was much longer than in iPS cells, where it persisted for a few days. The findings have been reported in Nature Communications.
This work might have a significant impact on gene-editing designs, suggested senior study author and Gladstone Senior Investigator Bruce Conklin, MD. "This could be an important safety consideration. If Cas9 hangs around longer, it has more chances to do its job and make on-target edits, which we want. But it also has more chances to make off-target edits, which we don't want. We will have to factor this in when designing therapies,” said Conklin.
After the DNA was cut by Cas9, the DNA sequences that were newly created by the editing tools were also very different. While many different sequences were generated through this approach in iPS cells, only a few of them were actually observed in neurons. This may mean that the outcome of CRISPR gene editing is far more predictable in non-dividing cells.
The team also found that CRISPR triggered the activity of some DNA repair genes in neurons that were once thought to be off-limits in non-dividing cells.
The researchers developed new methods for promoting certain results in neurons as well.
"Our ultimate goal is to precisely control the gene editing process to deliver life-changing therapies," noted Conklin. "And now, we have important new tools to make sure we get this right."
Sources: Gladstone Institutes, Nature Communications