Experiments using human embryos end up with loss of whole chromosomes

A new publication describes experiments using CRISPR/Cas9 gene scissors on human embryos. The aim of the experiments conducted in the US was to correct a mutated DNA sequence that causes a genetic disorder. This disorder can result in blindness (Retinitis pigmentosa). The gene scissors were supposed to cut the faulty gene sequence – and the expectation was that the fault in the genome would then be corrected via cell repair mechanisms. But this aim was not accomplished, as Testbiotech reports. Instead, either large parts or the whole of chromosome 6, where the gene is located, were lost. In addition, there were further unintended mutations in the target gene sequence.

As expected, the gene scissors cut both strands of the DNA at the target site. However, some of the cells were unable to repair the double-strand break correctly by re-joining the strands of DNA. In some embryos, either large parts of the chromosome or the whole chromosome were lost during subsequent cell divisions.

In addition, unintended effects were caused in another gene that is similar to the target gene but located on a different chromosome. Such unexpected activity of the gene scissors also led to some embryos losing this chromosome.

Loss of chromosomal regions can severely impact the development of embryos: cancer as well as organ malfunction may be the result, and in many cases the embryos will die before birth.

According to Testbiotech, there is currently a tendency to ethically questionable experiments being carried out with gene scissors on human embryos. At the same time, it is becoming clearer that applications of CRISPR/Cas9 are not as precise and safe as claimed. Several publications show that gene scissor applications in plants, animals and humans are flawed: the introduction of double-strand breaks very often facilitates the occurrence of unintended effects, e.g. the insertion of additional DNA sequences or genomic rearrangements at the target site. The target gene is also frequently mistaken for similar genes. Even the loss of larger chromosomal regions has been reported several times.

In 2020, the inventors of the gene scissor CRISPR/Cas were awarded the Nobel Prize for chemistry, despite a multitude of unanswered questions regarding risks and ethical problems associated with gene scissor applications. Currently, the evidence is mounting that the most frequently used gene scissor applications are not suitable for precise and safe interventions in the genome.

Source of comment: Testbiotech
https://www.testbiotech.org/en/news/crispr-gene-scissors-cause-chaos-genome
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Allele-specific chromosome removal after Cas9 cleavage in human embryos
Michael V. Zuccaro et al.
Cell, October 29, 2020. DOI: https://doi.org/10.1016/j.cell.2020.10.025
https://www.cell.com/cell/fulltext/S0092-8674(20)31389-1?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420313891%3Fshowall%3Dtrue

Highlights

• Cas9-mediated DSB induction and repair by end joining occurs within hours
• End joining provides an efficient way to restore reading frames without mosaicism
• Unrepaired DSBs persist through mitosis and result in frequent chromosome loss
• Off-target effects of Cas9 cause indels as well as chromosome loss

Summary

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. 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. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading 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. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.