Gene editing with CRISPR can lead to off-target mutations, but this appears to be less common because the enzyme cuts one DNA strand instead of two
July 1, 2022
A new form of genome editing technology CRISPR could provide a more accurate way to edit mutations that cause genetic diseases.The method was tested in Drosophila, fixing genetic mutations in a chromosome By using the equivalent chromosome inherited from the other parent as a template.
CRISPR typically works with a protein called Cas9, which acts as molecular scissors, cutting both strands of a DNA molecule at the site of a target sequence. This can allow new DNA sequences to be inserted between the cuts to replace the mutated gene.
However, this insertion is usually suitable for less than 10% cells And insertions can occur in incorrect or off-target regions of the genome.
Now, Ethan Bill and Annabel Guichard UC San Diego and their colleagues have developed a new form of CRISPR that more efficiently inserts the correct DNA sequence at the site of a mutation, reducing off-target effects.
“I was blown away,” Bill said. “Generally speaking, with existing CRISPR technology, you have to worry about about 1 percent of your edits being wrong or off-target. I’d say, with our system, it’s more like 1 in 10,000.”
The method uses a variant of the Cas9 enzyme called a nickase, which cuts only one strand of the DNA double helix. “We found that ‘gentlely’ cutting or cutting a DNA strand is more efficient than making a clean double-strand break,” Bier said.
The researchers tested the approach on fruit flies that had a mutation that made their eyes white instead of red. They found that the nickase system corrected eye color mutations in up to 65 percent of cells, making the fly’s eyes red. Standard CRISPR using Cas9 corrected the mutation in up to 30 percent of the cells, resulting in a small patch of red in each eye.
“It was an incredible moment. When we saw it right away, we knew we had found something absolutely amazing,” Guichard said.
The team didn’t introduce any extra pieces of DNA as cellular templates to correct the mutation on the chromosome, so the molecular machine had to use another chromosome — inherited from the other parent — as a template. The team was able to confirm that this was indeed the case.
It is generally considered impossible to perform DNA repair on one chromosome using another corresponding chromosome. But recent findings suggest that this occasionally occurs in specific, yet-to-be-identified circumstances.
“There is growing evidence that when you damage one chromosome in a mammalian cell, you somehow recruit the other chromosome. Then the damaged region gets a Band-Aid from the other chromosome,” Beer said .
“We really don’t understand what’s causing it. An exciting element of this work is that it opens up a pathway to discovering the entire suite of components responsible for this new class of repair.”
If it proves to work in humans, the approach could potentially fix any disease-associated genetic mutations that have healthy copies on matching chromosomes. This means it will not be able to repair the mutation on the X chromosome in boys, men and transgender women because they lack a second copy of the sex chromosome. It also doesn’t apply to people who have the exact same disease-associated mutation on both chromosomes in each parent.
Journal references: scientific progress, DOI: 10.1126/sciadv.abo072
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