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 is still a major concern.
Published July 1 in the journal Science Advances, a team of biologists from the University of California at San Diego that includes postdoctoral scholar Sitara Roy, specialist Annabel Guichard, and Professor Ethan Bier describes a new and safer approach which can correct genetic defects in the future. His strategy, which makes use of natural DNA repair machinery, provides a basis for new gene therapy strategies with the potential to cure a wide spectrum of genetic diseases.
In many cases, those with genetic disorders carry different mutations in the two copies of genes inherited from their parents. This means that often a mutation on one chromosome will have a functional sequence counterpart on the other chromosome. The researchers used CRISPR genetic editing tools to exploit this fact.
“The cell repair machinery can use the healthy variant to correct the defective mutation after cutting the mutant DNA,” said Guichard, the study’s lead author. “
Working on fruit flies, the researchers designed mutants that allow the visualization of this “homologous chromosome template repair” or HTR, by producing pigments in the eyes. These mutants initially had completely white eyes. But when the same flies expressed CRISPR components (a guide RNA plus Cas9), they showed large red spots on the eyes, a sign that the cell’s DNA repair machinery had managed to reverse the mutation using DNA. functional of the other chromosome.
They then tested their new system with Cas9 variants known as “nickases” that targeted only one strand of DNA instead of both. Surprisingly, the authors found that these spots also resulted in a high-level restoration of red eye color almost the same as 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 in the genome (the called off-target mutations). “I couldn’t believe how well nickase worked; it was completely unexpected,” 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 still don’t know how this process will translate into human cells and whether we can apply it to any gene,” Guichard said. “Some adjustment may be needed to obtain efficient HTR for mutations that cause diseases carried by human chromosomes.”
The new research expands the group’s previous successes in precision editing with “allelic units,” which extend the principles of genetic units 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 gen.
A key feature of the team’s research is that its nickase-based system causes far fewer mutations inside and outside the target, as is known to happen with more traditional Cas9-based CRISPR editions. They also say that a slow and continuous delivery of nickase components over several days can be more beneficial than punctual deliveries.
“Another notable advantage of this approach is its simplicity,” Bier said. “It depends on very few components and the DNA peaks are ‘soft’, unlike Cas9, which produces complete DNA breaks often accompanied by mutations.”
“If the frequency of these events could be increased, either by promoting interhomologous mating or by optimizing nick-specific repair processes, these strategies could be leveraged to correct numerous mutations that cause dominant or trans-heterozygous diseases,” he said. Roy.
Complete list of authors of the document Science Advances: Sitara Roy, Sara Sanz Juste, Marketta Sneider, Ankush Auradkar, Carissa Klanseck, Zhiqian Li, Alison Henrique Ferreira Julio, Victor Lopez del Amo, Ethan Bier and Annabel Guichard.
Research support was provided by the National Institutes of Health (grant R01 GM117321), a Paul G. Allen Frontiers Group Distinguished Investigators Award, and a gift from Tata Trusts in India to the Tata Institute for Genetics and Society (TIGS ) -UC San Diego and TIGS India.
Competitive Interest Note: Bier has a stake in two companies it co-founded: Synbal Inc. and Agragene, Inc., who may benefit from the research results. He is also a member of the board of directors of Synbal and the scientific advisory board of both companies.