Genome editing (also called gene editing) is a group of technologies that give scientists the ability to alter an organism’s DNA. These techniques allow genetic material to be added, removed, or altered at specific locations in the genome. Several methods of genome editing have been developed. One known one is called CRISPR-Cas9, which is short for CRISPR-related short palindromic repeat and protein-related 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other genome-editing methods.
Ten years ago this week, Jennifer Doudna and her colleagues published the results of a test-tube experiment on bacterial genes, titled, “An RNA-guided, programmable double-stranded DNA exonuclease in adaptive bacterial immunity.” This discovery led to a new type of genome editing, now known as CRISPR.
CRISPR-Cas9 — short for clusters of regularly spaced short palindromic repeats and CRISPR-related protein 9 — enables rapid editing of the genome. Over the past decade, the landscape of diseases under study has changed dramatically. Doctors use CRISPR technology to modify genes that cause genetic diseases, and since cancer biologists discovered that changes in DNA cause cancer, they have been using the method to correct these changes by manipulating the DNA.
Since scientists realized that changes in DNA cause cancer, they have been looking for an easy way to correct those changes by manipulating the DNA.
Now, a team of biologists at the University of California San Diego, which includes postdoctoral scientist Sitara Roy, specialist Annabelle Guichard and Professor Ethan Pierre, has described a new and safer method using the natural DNA repair machinery that may correct genetic defects in the future.
This approach provides a basis for new gene therapy strategies that have the potential to treat a wide range of genetic diseases.
As in many cases, those with genetic disorders carry distinct mutations in two copies of genes inherited from their parents. This can mean that a mutation on one chromosome will have a functional sequence counterpart on the other chromosome. The researchers used CRISPR gene-editing tools to “exploit” this fact.
“A healthy variant can be used by the cell’s repair machinery to correct the defective mutation after cutting the mutated DNA,” Guichard, senior author of the study, said in a statement. “Remarkably, this can be achieved most effectively with a simple, harmless blow.”
The researchers worked with fruit flies. They engineered mutants that allowed such “homologous chromosome template repair,” or HTR, to be visualized by producing pigments in their eyes. Initially, these mutants were distinguished by completely white eyes. But when the same flies expressed components of CRISPR (guide RNA plus Cas9), they showed large red spots across their eyes.
This was a sign that the cell’s DNA repair machinery had successfully reversed the mutation by using functional DNA from the other chromosome.
The scientists then tested their new system using Cas9 variants known as “Nickases” that targeted just one strand of DNA instead of both.
Surprisingly, the authors found that such nicks also resulted in high-level restoration of red-eye color nearly on par with normal (non-mutated) healthy flies. A 50–70 percent repair success rate was found with nicase, compared to only 20–30 percent for the Cas9 double-stranded cut, which also produces frequent mutations and targets other sites throughout the genome.
“I couldn’t believe how well the poke worked—it was totally unexpected,” said Roy, lead author of the study.
“We don’t know yet how this process will translate into human cells and whether we can apply it to any gene,” Guichard said. “Some modification may be required to obtain an effective HTR for disease-causing mutations carried by human chromosomes.”
“Another notable advantage of this approach is its simplicity,” said Pierre. “It relies on very few components, ‘soft’ DNA points, unlike Cas9, which produces complete DNA breaks often accompanied by mutations.”
“If the frequency of such events can be increased either by enhancing coupling between homologues or by improving nicotine-specific repair processes, then such strategies could be harnessed to correct many disease-causing dominant or heterozygous mutations,” Roy said.
The new system’s “ingenuity” could serve as a model for repairing genetic mutations in mammals.