Gene editing using CRISPR can cause off-target mutations, which do not appear to occur less often with enzymes that cleave one strand of DNA rather than both.
July 1, 2022
A new form of genome editing technology CRISPR It may provide a more accurate way to edit the mutations that cause a genetic disease.The approach tested in Drosophila corrects a genetic mutation in one copy Chromosome Use equivalent chromosomes inherited from other parents as templates.
CRISPR usually works with a protein called Cas9. It acts as a molecule scissors, cutting two strands of a DNA molecule at the site of the target sequence. This allows a new DNA sequence to be inserted between cuts to replace the mutated gene.
However, this insertion is usually Less than 10 percent of cells Insertions can occur in false or off-target regions of the genome.
now, Ethan Beer When Annabel Guichard The University of California, San Diego and colleagues have developed a new form of CRISPR that can more efficiently insert the correct DNA sequence at the mutation site and reduce off-target effects.
“I was surprised,” says Bier. “In general, with existing CRISPR technology, you have to worry about about 1% of edits being mistaken or off-target. For our system, it’s like 1 / 10,000.”
This method uses a variant of the Cas9 enzyme called nickase. It cuts only one strand of the DNA double helix. “We have found that nicking or cutting a single strand of DNA“ softly ”is even more efficient than performing a clean double-strand break,” says Bier.
Researchers tested the approach with fruit flies with mutations that turn the eyes white instead of red. They found that the nickase system corrects eye color mutations in up to 65% of cells, giving flies red eyes. Standard CRISPR using Cas9 corrected mutations in up to 30% of cells, with small red spots on each eye.
“It was a really great moment. When we saw it right away, we knew we had found something absolutely amazing,” says Guichard.
The molecular machine must have used other chromosomes inherited from other parents as templates, as the team did not introduce extra pieces of DNA as a cell template to correct mutations on the chromosome. The team was able to confirm that this was the case.
DNA repair of one chromosome using the other corresponding chromosome was not generally considered possible. However, recent findings suggest that this can occasionally occur under certain undefined circumstances.
“There is accumulating evidence that damaging one chromosome in a mammalian cell somehow mobilizes the other. Next, the broken region gets Band-Aid from the other chromosome.” Bier says.
“We really don’t understand if we’re responsible for doing that. One of the exciting elements of this task is paving the way for discovering the entire set of components responsible for this new category of repairs. is.”
If it proves to work in people, this approach has the potential to repair disease-related genetic mutations that have healthy copies on matching chromosomes. This means that the X chromosome mutations in boys, men and transgender women who do not have a second copy of this sex chromosome cannot be corrected. It also does not work for people who have mutations associated with the exact same disease on both chromosomes of each parent.
Journal reference: Science Advances, DOI: 10.1126 /sciadv.abo072
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CRISPR: A “softer” form of gene editing technology may be more accurate
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