Curing debilitating genetic diseases is one of the great challenges of modern medicine. Over the past decade, the development of CRISPR technologies and advances in genetic research have brought new hope to patients and their families, although the safety of these new methods remains a major concern.
Publication in the journal Scientists progress, a team of biologists from the University of California, San Diego that includes postdoctoral researcher Sitara Roy, specialist Annabel Guichard and Professor Ethan Bier describes a new, safer approach that could correct genetic defects in the future. Their strategy, which uses the natural DNA repair machinery, provides a basis for new gene therapy strategies with the potential to cure a wide range of genetic diseases.
In many cases, people with genetic disorders carry distinct mutations in both copies of genes inherited from their parents. This means that often a mutation on one chromosome will have a homologous functional sequence on the other chromosome. The researchers used CRISPR gene-editing tools to exploit this fact.
“The healthy variant can be used by the cell’s repair machinery to correct the faulty mutation after cutting the mutant DNA,” said Guichard, the study’s lead author, “remarkably, this can still be achieved more effectively by a simple harmless gash.”
Working on fruit flies, the researchers engineered mutants that allowed this “homologous chromosome repair,” or HTR, to be visualized through the production of pigment in their eyes. These mutants initially featured all-white eyes. But when the same flies expressed CRISPR components (a guide RNA plus Cas9), they displayed large red spots on their eyes, a sign that the cell’s DNA repair machinery had managed to reverse the mutation using the Functional DNA from the other chromosome.
They then tested their new system with Cas9 variants known as “nickases” that targeted a single strand of DNA instead of both. Surprisingly, the authors found that such nicks also resulted in a high-level restoration of red-eye color almost on par with normal (non-mutated) healthy flies. They found a 50-70% repair success rate with nickase, compared to only 20-30% in the Cas9 double-stranded cut, which also generates frequent mutations and targets other sites throughout the genome (mutations say off target). “I couldn’t believe how well the nickase worked – it was completely unplanned,” said Roy, the study’s lead author. The versatility of the new system could serve as a model for fixing genetic mutations in mammals, the researchers noted.
“We don’t yet know how this process will translate into human cells and whether we can apply it to any gene,” Guichard said. “Some adjustments may be needed to obtain an effective HTR for pathogenic mutations carried by human chromosomes.”
The new research extends the band’s previous accomplishments in precision editing with “allelic urgeswhich develops the principles of gene drives with a guide RNA that directs the CRISPR system to cut unwanted variants of a gene and replace them with a preferred version of the gene.
A key feature of the team’s research is that their nickase-based system causes far fewer on- and off-target mutations, as occurs with more traditional Cas9-based CRISPR edits. They also say that slow, continuous delivery of nickase components over several days may prove more beneficial than one-time deliveries.
“Another notable advantage of this approach is its simplicity,” Bier said. “It relies on very few components and DNA nicks are ‘soft’, unlike Cas9, which produces complete DNA breaks often accompanied by mutations.”
“If the frequency of such events could be increased either by promoting interhomologous pairing or by optimizing nick-specific repair processes, such strategies could be exploited to correct many dominant or trans-heterozygous pathogenic mutations,” said Roy.