Gene editing with CRISPR can cause off-target mutations, but this seems to happen less often with an enzyme that cuts one of the strands of DNA instead of both
Life
1 July 2022
CRISPR uses an enzyme called Cas9 to cut DNA at precise locations
A new form of the genome-editing technique CRISPR could provide a more accurate way to edit mutations that cause genetic diseases. The approach, which was tested in fruit flies, fixes a genetic mutation on one copy of a chromosome by using the equivalent chromosome – inherited from the other parent – as a template.
CRISPR usually works with a protein called Cas9, which acts as molecular scissors to cut through the two strands of a DNA molecule at the site of a targeted sequence. This can allow new DNA sequences to be inserted between the cuts to replace the mutated gene.
However, this insertion usually works for less than 10 per cent of cells and insertions can occur in incorrect, or off-target, regions of the genome.
Now, Ethan Bier and Annabel Guichard at the University of California, San Diego, and their colleagues have developed a new form of CRISPR that can more efficiently insert correct DNA sequences at the site of a mutation, with fewer off-target effects.
“I was blown away,” says Bier. “In general, with existing CRISPR techniques, you have to worry about roughly 1 per cent of edits being mistakes or off-target. I would say that, in the case of our system, it would be more like 1 in 10,000.”
The method uses a variant of the Cas9 enzyme called a nickase, which only cuts one strand of the DNA double helix. “We found that ‘softly’ nicking, or cutting, one strand of the DNA is even more efficient than making a clean double-stranded break,” says Bier.
The researchers tested the approach in fruit flies that had a mutation that turned their eyes white instead of red. They found that the nickase system corrected the eye colour mutation in up to 65 per cent of cells, giving the flies red eyes. Standard CRISPR using Cas9 corrected the mutation in up to 30 per cent of cells, causing each eye to have a small patch of red.
“It was a truly incredible moment. We knew we had found something absolutely amazing when we saw that right away,” says Guichard.
The team didn’t introduce any extra pieces of DNA as a template for the cell to correct the mutation on the chromosome, so the molecular machinery must have used the other chromosome – inherited from the other parent – as a template. The team was able to confirm this was the case.
DNA repair of one chromosome using the other corresponding chromosome was generally not thought to be possible. But recent findings suggest that this can occasionally occur under specific circumstances that have yet to be defined.
“There’s accumulating evidence that when you create damage to one chromosome in a mammalian cell, then that somehow recruits the other chromosome. Then the region that’s broken gets the Band-Aid from the other chromosome,” says Bier.
“We don’t really understand what is responsible for doing that. One of the exciting elements of the work is that it opens up an avenue of discovering the whole set of components that are responsible for this new category of repair.”
If it is proven to work in people, the approach could potentially repair any disease-associated genetic mutations that have a healthy copy on the matching chromosome. This means it won’t be able to fix mutations on the X chromosome in boys, men and transgender women, who lack a second copy of this sex chromosome. It also won’t work for people with the exact same disease-linked mutation on both chromosomes from each parent.
Journal reference: Science Advances, DOI: 10.1126/sciadv.abo072
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