Responsible scientists insist that the genome editing of human embryos presents unacceptable risks, if only because it can bring about subtle—but still deleterious—off-target effects. One wonders what these scientists would say if they learned that gene editing could wreak harms that were anything but subtle, harms as catastrophic as the loss of an entire chromosome.

Such large-scale damage was found in a study that analyzed human embryos that had been edited with CRISPR-Cas9. The study, from Columbia University Irving Medical Center, showed that applying CRISPR-Cas9 to repair a blindness-causing gene early in the development of a human embryo often eliminates an entire chromosome or a large section of it.

Details of this work appeared recently in the journal Cell, in an article titled, “Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.” The article describes how Columbia University scientists led by Dieter Egli, PhD, tested CRISPR-Cas9 genome editing’s effects on early-stage human embryos carrying a mutation in a gene called EYS (eyes shut homolog), which causes hereditary blindness.

“Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness,” the article’s authors wrote. “We show that the most common repair outcome is microhomology-mediated end joining, which [leads to] embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms.”

Besides observing that on-target cleavage may result in unrepaired DSBs that persist through mitosis and result in chromosome loss, the Columbia scientists also saw that Cas9 off-target cleavage may result in chromosomal losses and hemizygous indels because of cleavage of both alleles.

This graphical abstract from a paper published in Cell (DOI: 10.1016/j.cell.2020.10.025) shows repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus.

“Our study shows that CRISPR/Cas9 is not yet ready for clinical use to correct mutations at this stage of human development,” said Egli, the study’s senior author and assistant professor of developmental cell biology in the department of pediatrics at Columbia University.

The first use of CRISPR in human embryos was reported in 2015. Just a few years later, in 2018, He Jiankui claimed to have performed the procedure in a pair of twins, eliciting a firestorm of condemnation from scientists and government officials worldwide.

He Jiankui’s “CRISPR babies” announcement occurred after a 2017 study that had reported the successful correction of a heart disease–causing mutation in normal human embryos using CRISPR. This study, however, may have been misinterpreted.

According to the new study by Egli and colleagues, the 2017 study may indicate that instead of being corrected, the chromosome carrying the heart disease–causing mutation may have been lost altogether.

“If our results had been known two years ago, I doubt that anyone would have gone ahead with an attempt to use CRISPR to edit a gene in a human embryo in the clinic,” Egli asserted. “Our hope is that these cautionary findings should discourage premature clinical application of this important technology, but can also guide responsible research to achieve its ultimate safe and effective use.”

“We know from previous studies in differentiated human cells and in mice that a break in the DNA results in mostly two outcomes: precise repair or small local changes,” added Michael Zuccaro, a PhD candidate at Columbia University Vagelos College of Physicians and Surgeons and co-lead author of the paper. “At the EYS gene, these changes can yield a functional gene, though it is not a perfect repair.”

When Zuccaro, Egli, and their colleagues looked at the entire genomes of the modified embryos in this study, they identified another outcome. “We learned,” said Zuccar, “that in human embryonic cells, a single break in the DNA can result in a third outcome, the loss of an entire chromosome or sometimes a large segment of that chromosome, and this loss of the chromosome is very frequent.”

Egli and colleagues noted that their findings “reveal significant challenges for mutation correction in human embryos.”

“Our results,” they concluded, “serve as a cautionary note for the use of induced DSBs in editing the genome for clinical use. Chromosomal material might be lost because of on-target and off-target activity of Cas9 and result in aneuploidy and developmental abnormalities. Upon further investigation, alternatives that do not require a DSB for mutation correction, such as base or prime editing, might be preferable to precisely correct mutations in the germline.”

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