This means that normally, a mutation on one chromosome will have a functional sequence copy on the other. The researchers used the gene editing tool CRISPR to exploit this fact.
“The healthy variant can be used by the cell’s repair machinery to repair the faulty mutation after cutting the mutant DNA,” said Guichard, senior author of the study. ”
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Working in fruit flies, the researchers engineered mutants that allow visualization of “homologous chromosomal patterned repair,” or HTR, by producing pigments. in their eyes. Such mutants originally had completely white eyes. But when the same flies expressed CRISPR components (a guide RNA plus Cas9), they displayed large red patches on their eyes, a sign that the cell’s DNA repair machinery had matured. work in reversing the mutation using functional DNA from another chromosome.
CRISPR Gene Editing
They then tested their new system with so-called “nickname” Cas9 variants that target just one strand of DNA instead of both. Surprisingly, the authors found that such cracks also increased red-eye color recovery to a high degree nearly equal to that of normal (non-mutated) healthy flies.
They found a 50-70% successful repair rate with nickase compared to only 20-30% in double-stranded Cas9, which also generates mutations frequently and targets other sites throughout the genome ( called off-target mutations). “I couldn’t believe how well nickase worked – it was completely unforeseen,” said Roy, the study’s lead author. The researchers note that the new system’s flexibility could serve as a model for repairing genetic mutations in mammals.
“We still don’t know how this process will translate to human cells, and if we can apply it to any gene,” says Guichard. “Several adjustments may be needed to obtain an effective HTR for carrier-borne disease-causing mutations.”
The new study expands on the team’s previous achievements in precision editing with “allele drives,” expanding on the principles of gene drive with a guide RNA that directs the CRISPR system to cut unwanted variants. wants of genes and replace them with a preferred version. of genes.
A key feature of the team’s study is that their nickase-based system induces much fewer on- and off-target mutations, as is known to occur with more traditional Cas9-based CRISPR edits. They also say that a slow, continuous delivery of nickase ingredients over several days may be more beneficial than a one-time delivery.
“Another notable advantage of this approach is its simplicity,” says Bier. “It depends on very few components and the ‘soft’ DNA slots, unlike Cas9, produce full DNA breaks that are often accompanied by mutations.”
“If the frequency of such events can be increased by promoting cell-to-cell coupling or by optimizing specific repair processes, then such strategies could be increased,” says Roy. can be used to repair many dominant or transgenic mutations,” says Roy.
Source: Eurekalert
