Jennifer Doudna knew how powerful the CRISPR-Cas9 gene editing system was/could be when she invented it in 2012. It’s one of the reasons why she called for an immediate moratorium on the method, ensuring ethics and safety were put before advancement.
Eleven years later, CRISPR/Cas9 has helped scientists make incredible strides in disease research, finding applications in everything and anything—beside embryonic research. It is still illegal in almost all countries to use the system on an embryo intended to establish pregnancy. (Although that did not stop Chinese scientist He Jiankui. He used CRISPR/Cas9 to help make the world’s first genetically edited babies—twin girls born in November 2018. A third baby, carried by a second woman, was reportedly born in Summer 2019. He was subsequently sentenced to three years in prison.)
New research by scientists at the University of Oxford (UK) supports Doudna’s inclination for more research on the CRISPR/Cas9 system before use on embryos.
“Our results show that the use of CRISPR-Cas9 in early human embryos carries significant risks,” Nada Kubikova, a reproductive biologist at Oxford, said during the 39th annual meeting of the European Society of Human Reproduction and Embryology. “We have found that the DNA of embryo cells can be targeted with high efficiency, but unfortunately this rarely leads to the sort of changes needed to correct a defective gene. More often, the strand of DNA is permanently broken, which could potentially lead to additional genetic abnormalities in the embryo.”
Two types of DNA repair
All the cells of the body have highly efficient mechanisms for repairing damage affecting their DNA. The most common way, known as non-homologous end joining, is for cells to reconnect the two ends of the DNA strands after a double-stand break. However, when this happens, it is common for a few letters of genetic code to be deleted or duplicated at the site where the strands are reattached. This can disrupt genes and does not allow mutations to be corrected.
A second way for cells to repair a break in DNA is by using an intact copy of the affected area as a template, copying it and replacing the damaged area. Rather than copying a mutation, researchers can intervene to supply cells pieces of DNA containing slightly altered, normal DNA sequences. Cells can then use these templates to repair breaks, removing the broken piece of DNA and copying the rest of the supplied sequence at the same time. This is known as homology-directed repair, and is the process required for correcting a mutation.
Repairing breaks
In an ethically approved study, Kubikova and her colleagues fertilized donated eggs with donated sperm to create 84 embryos. In 33 of the embryos, they used CRISPR-Cas9 to introduce DNA double-strand breaks. The remaining 51 embryos were kept as controls.
According to the research, presented at the conference, the team detected alterations at the targeted DNA sites in 24 out of 25 embryos, indicating that CRISPR is highly efficient in the cells of human embryos. However, only 9% of targeted sites were repaired using the clinically useful process of homology-directed repair. Fifty-one percent of broken DNA strands underwent non-homologous end joining, producing mutations where the strands were reconnected.
The remaining 40% of broken DNA strands failed to be repaired. The unrepaired breaks in the DNA strands eventually led to large pieces of chromosome, which extend from the site of the break to the end of the chromosome, being lost or duplicated. Abnormalities of this type affect the viability of embryos and if affected embryos were transferred to the uterus and produced a baby, they would carry a risk of serious congenital abnormalities, the researchers said.
“Non-homologous end joining introduces additional mutations rather than correcting existing ones,” said Kubikova. “This would be a challenge if there were attempts to use CRISPR-Cas9 to correct inherited disorders in human embryos, as it suggests that most times when it is attempted, it will not be successful.”
While the study results caution against the use of genome editing in human embryos, there were some positive findings, suggesting that risks can be lowered by modifying the way in which genome editing is undertaken. Additionally, the findings have broader implications for IVF.
“On average, only about a quarter of the embryos created using IVF succeed in producing a baby. Half of them stop developing in the laboratory before they can be transferred to the womb,” said Kubikova. “The inability of embryos to efficiently repair DNA damage, revealed by this study, may explain why some IVF embryos fail to develop. This understanding may lead to improved IVF treatments.”
Now, the researchers will look for new ways to protect early embryos from DNA damage, which again could have significance for fertility treatments. The team also plans to explore more gentle methods of gene editing that avoid breakage of the DNA strands, which embryos might find easier to cope with.