A softer form of CRISPR may be able to edit genes more precisely.

 Off-target mutations can occur with CRISPR gene editing, but this appears to be less common with an enzyme that cuts one of the DNA strands rather than both.


A new type of genome editing technique CRISPR may provide a more precise method of editing mutations that cause genetic diseases. The method, which was tested in fruit flies, corrects a genetic mutation on one copy of a chromosome by using the equivalent chromosome from the other parent as a template.

CRISPR is typically used in conjunction with the Cas9 protein, which acts like molecular scissors to cut through the two strands of a DNA molecule at the site of a targeted sequence. This allows for the insertion of new DNA sequences between the cuts to replace the mutated gene.

However, less than 10% of cells benefit from this insertion, and insertions can occur in incorrect, or off-target, regions of the genome.

Ethan Bier and Annabel Guichard of the University of California, San Diego, and their colleagues have now developed a new type of CRISPR that can insert correct DNA sequences at the site of a mutation more efficiently and with fewer off-target effects.

"I was blown away," Bier says. "In general, with existing CRISPR techniques, you have to be concerned about roughly 1% of edits being incorrect or off-target." In the case of our system, I'd say it's closer to one in 10,000."

The method employs a nickase variant of the Cas9 enzyme, which only cuts one strand of the DNA double helix. "We discovered that softly nicking, or cutting, one strand of DNA is more efficient than making a clean double-stranded break," Bier explains.

The approach was tested in fruit flies with a mutation that turned their eyes white instead of red. They discovered that the nickase system corrected the eye color mutation in up to 66% of cells, resulting in red eyes in the flies. Standard CRISPR using Cas9 corrected the mutation in up to 30% of cells, resulting in a small patch of red in each eye.

"It was an incredible moment." "When we saw that right away, we knew we'd found something absolutely incredible," Guichard says.

Because the researchers did not use any extra pieces of DNA as a template for the cell to correct the chromosome mutation, the molecular machinery must have used the other chromosome - inherited from the other parent - as a template. The team was able to confirm that this was correct.

It was previously thought that DNA repair of one chromosome using the other corresponding chromosome was not possible. However, new research suggests that this can happen on occasion under specific circumstances that have yet to be defined.

"There is mounting evidence that when one chromosome in a mammalian cell is damaged, the other chromosome is recruited." "Then the broken region gets a Band-Aid from the other chromosome," Bier explains.

"We don't know who or what is to blame for this." One of the most exciting aspects of the work is that it opens up the possibility of discovering the entire set of components responsible for this new category of repair."

If it is shown to be effective in humans, the method could potentially repair any disease-associated genetic mutations that have a healthy copy on the matching chromosome. This means that it will be unable to correct X chromosome mutations in boys, men, and transgender women who lack a second copy of this sex chromosome. It will also not work for people who have the same disease-linked mutation from both parents on both chromosomes.

Reference: Science Advances, DOI: 10.1126/sciadv.abo072


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